An Intrinsic Host Defense against HSV-1 Relies on the Activation of Xenophagy with the Active Clearance of Autophagic Receptors.

Autophagy engulfs cellular components in double-membrane-bound autophagosomes for clearance and recycling after fusion with lysosomes. Thus, autophagy is a key process for maintaining proteostasis and a powerful cell-intrinsic host defense mechanism, protecting cells against pathogens by targeting them through a specific form of selective autophagy known as xenophagy. In this context, ubiquitination acts as a signal of recognition of the cargoes for autophagic receptors, which direct them towards autophagosomes for subsequent breakdown. Nevertheless, autophagy can carry out a dual role since numerous viruses including members of the Orthoherpesviridae family can either inhibit or exploit autophagy for its own benefit and to replicate within host cells. There is growing evidence that Herpes simplex virus type 1 (HSV-1), a highly prevalent human pathogen that infects epidermal keratinocytes and sensitive neurons, is capable of negatively modulating autophagy. Since the effects of HSV-1 infection on autophagic receptors have been poorly explored, this study aims to understand the consequences of HSV-1 productive infection on the levels of the major autophagic receptors involved in xenophagy, key proteins in the recruitment of intracellular pathogens into autophagosomes. We found that productive HSV-1 infection in human neuroglioma cells and keratinocytes causes a reduction in the total levels of Ub conjugates and decreases protein levels of autophagic receptors, including SQSTM1/p62, OPTN1, NBR1, and NDP52, a phenotype that is also accompanied by reduced levels of LC3-I and LC3-II, which interact directly with autophagic receptors. Mechanistically, we show these phenotypes are the result of xenophagy activation in the early stages of productive HSV-1 infection to limit virus replication, thereby reducing progeny HSV-1 yield. Additionally, we found that the removal of the tegument HSV-1 protein US11, a recognized viral factor that counteracts autophagy in host cells, enhances the clearance of autophagic receptors, with a significant reduction in the progeny HSV-1 yield. Moreover, the removal of US11 increases the ubiquitination of SQSTM1/p62, indicating that US11 slows down the autophagy turnover of autophagy receptors. Overall, our findings suggest that xenophagy is a potent host defense against HSV-1 replication and reveals the role of the autophagic receptors in the delivery of HSV-1 to clearance via xenophagy.

Physiological and Biochemical Aspects of Silicon-Mediated Resistance in Maize against Maydis Leaf Blight.

Maydis leaf blight (MLB), caused by the necrotrophic fungus Bipolaris maydis, has caused considerable yield losses in maize production. The hypothesis that maize plants with higher foliar silicon (Si) concentration can be more resistant against MLB was investigated in this study. This goal was achieved through an in-depth analysis of the photosynthetic apparatus (parameters of leaf gas exchange chlorophyll (Chl) a fluorescence and photosynthetic pigments) changes in activities of defense and antioxidative enzymes in leaves of maize plants with (+Si; 2 mM) and without (-Si; 0 mM) Si supplied, as well as challenged and not with B. maydis. The +Si plants showed reduced MLB symptoms (smaller lesions and lower disease severity) due to higher foliar Si concentration and less production of malondialdehyde, hydrogen peroxide, and radical anion superoxide compared to -Si plants. Higher values for leaf gas exchange (rate of net CO2 assimilation, stomatal conductance to water vapor, and transpiration rate) and Chl a fluorescence (variable-to-maximum Chl a fluorescence ratio, photochemical yield, and yield for dissipation by downregulation) parameters along with preserved pool of chlorophyll a+b and carotenoids were noticed for infected +Si plants compared to infected -Si plants. Activities of defense (chitinase, ß-1,3-glucanase, phenylalanine ammonia-lyase, polyphenoloxidase, peroxidase, and lipoxygenase) and antioxidative (ascorbate peroxidase, catalase, superoxide dismutase, and glutathione reductase) enzymes were higher for infected +Si plants compared to infected -Si plants. Collectively, this study highlights the importance of using Si to boost maize resistance against MLB considering the more operative defense reactions and the robustness of the antioxidative metabolism of plants along with the preservation of their photosynthetic apparatus.

Altered purinergic P2X7 and A2B receptors signaling limits macrophage-mediated host defense in schistosomiasis.

BACKGROUND: The occurrence of co-infections during schistosomiasis, a neglected tropical disease, with other parasites have been reported suggesting an impaired host immune defense. Macrophage purinergic P2X7 receptor (P2X7R) play an important role against intracellular pathogens. Therefore, we investigated the P2X7R-mediated phagocytosis and killing capacity of Leishmania amazonensis by macrophages during schistosomiasis in vitro and in vivo. METHODS: Swiss and C57BL/6 (Wild type) and P2X7R-/- were randomized in two groups: control (uninfected) and Schistosoma mansoni-infected. Alternatively, control Swiss and S. mansoni-infected mice were also infected with L. amazonensis. RESULTS: The pre-treatment of macrophages with the P2X7R antagonist (A74003) or TGF-ß reduced the phagocytosis index, mimicking the phenotype of cells from S. mansoni-infected mice and P2X7R-/- mice. Apyrase also reduced the phagocytosis index corroborating the role of ATP to macrophage activation. Moreover, l-arginine-nitric oxide pathway was compromised, which could explain the reduced killing capacity in response to ATP in vitro and in vivo. We found an increased extracellular nucleotide (ATP, ADP and AMP) hydrolysis along with an increased frequency of F4/80+ CD39+ macrophages from the S. mansoni-infected group. Moreover, the content of adenosine in the cell supernatant was higher in the S. mansoni-infected group in relation to controls. Schistosomiasis also increased the expression of macrophage adenosine A2BR. In good accordance, both ADA and the selective A2BR antagonist restored the phagocytosis index of macrophages from S. mansoni-infected group. CONCLUSIONS: Altogether, the altered P2X7R and A2BR signaling limits the role of macrophages to host defense against L. amazonensis during schistosomiasis, potentially contributing to the pathophysiology and clinically relevant co-infections.

Antimicrobial peptides´ immune modulation role in intracellular bacterial infection.

Intracellular bacteria cause a wide range of diseases, and their intracellular lifestyle makes infections difficult to resolve. Furthermore, standard therapy antibiotics are often unable to eliminate the infection because they have poor cellular uptake and do not reach the concentrations needed to kill bacteria. In this context, antimicrobial peptides (AMPs) are a promising therapeutic approach. AMPs are short cationic peptides. They are essential components of the innate immune response and important candidates for therapy due to their bactericidal properties and ability to modulate host immune responses. AMPs control infections through their diverse immunomodulatory effects stimulating and/or boosting immune responses. This review focuses on AMPs described to treat intracellular bacterial infections and the known immune mechanisms they influence.

Peptidomic analysis of the host-defense peptides in skin secretions of the Amazon River frog Lithobates palmipes (Ranidae).

Skin secretions of certain frog species represent a source of host-defense peptides (HDPs) with therapeutic potential and their primary structures provide insight into taxonomic and phylogenetic relationships. Peptidomic analysis was used to characterize the HDPs in norepinephrine-stimulated skin secretions from the Amazon River frog Lithobates palmipes (Ranidae) collected in Trinidad. A total of ten peptides were purified and identified on the basis of amino acid similarity as belonging to the ranatuerin-2 family (ranatuerin-2PMa, -2PMb, -2PMc, and-2PMd), the brevinin-1 family (brevinin-1PMa, -1PMb, -1PMc and des(8-14)brevinin-1PMa) and the temporin family (temporin-PMa in C-terminally amidated and non-amidated forms). Deletion of the sequence VAAKVLP from brevinin-1PMa (FLPLIAGVAAKVLPKIFCAISKKC) in des[(8-14)brevinin-1PMa resulted in a 10-fold decrease in potency against Staphylococcus aureus (MIC = 31 µM compared with 3 µM) and a > 50-fold decrease in hemolytic activity but potency against Echerichia coli was maintained (MIC = 62.5 µM compared with 50 µM). Temporin-PMa (FLPFLGKLLSGIF.NH2) inhibited growth of S. aureus (MIC = 16 µM) but the non-amidated form of the peptide lacked antimicrobial activity. Cladistic analysis based upon the primary structures of ranaturerin-2 peptides supports the division of New World frogs of the family Ranidae into the genera Lithobates and Rana. A sister-group relationship between L. palmipes and Warszewitsch's frog Lithobates warszewitschii is indicated within a clade that includes the Tarahumara frog Lithobates tarahumarae. The study has provided further evidence that peptidomic analysis of HDPs in frog skin secretions is a valuable approach to elucidation of the evolutionary history of species within a particular genus.

Antimicrobial Spectrum of Activity and Mechanism of Action of Linear Alpha-Helical Peptides Inspired by Shrimp Anti-Lipopolysaccharide Factors.

Shrimp antilipopolysaccharide factors (ALFs) form a multifunctional and diverse family of antimicrobial host defense peptides (AMPs) composed of seven members (groups A to G), which differ in terms of their primary structure and biochemical properties. They are amphipathic peptides with two conserved cysteine residues stabilizing a central ß-hairpin that is understood to be the core region for their biological activities. In this study, we synthetized three linear (cysteine-free) peptides based on the amino acid sequence of the central ß-hairpin of the newly identified shrimp (Litopenaeus vannamei) ALFs from groups E to G. Unlike whole mature ALFs, the ALF-derived peptides exhibited an α-helix secondary structure. In vitro assays revealed that the synthetic peptides display a broad spectrum of activity against both Gram-positive and Gram-negative bacteria and fungi but not against the protozoan parasites Trypanosoma cruzi and Leishmania (L.) infantum. Remarkably, they displayed synergistic effects and showed the ability to permeabilize bacterial membranes, a mechanism of action of classical AMPs. Having shown low cytotoxicity to THP-1 human cells and being active against clinical multiresistant bacterial isolates, these nature-inspired peptides represent an interesting class of bioactive molecules with biotechnological potential for the development of novel therapeutics in medical sciences.

On the wave of the crustin antimicrobial peptide family: From sequence diversity to function.

Crustins represent the largest and most diverse family of antimicrobial peptides (AMPs) found in crustaceans. They are classically defined as disulfide-rich peptides/polypeptides holding a typical Whey Acidic Protein (WAP) domain at the C-terminal end. This WAP domain has eight cysteine residues forming a tightly packed structure, the four-disulfide core (4DSC) motif, that is also found in other proteins displaying protease inhibitory properties. Crustins are highly diverse in terms of primary structure, size and biochemical features, thus exhibiting a series of biological functions beyond their antimicrobial properties. In order to better categorize the distinct crustin members, different classification systems have been proposed. In this review, we discuss the current classification systems and explore the biological implication of the impressive molecular diversity of this unique AMP family. We also summarize the recent findings on the role of these effectors in crustacean immunity and homeostasis as well as in host-microbe interactions.

Time Course RNA-seq Reveals Soybean Responses against Root-Lesion Nematode and Resistance Players.

Pratylenchus brachyurus causes serious damage to soybean production and other crops worldwide. Plant molecular responses to RLN infection remain largely unknown and no resistance genes have been identified in soybean. In this study, we analyzed molecular responses to RLN infection in moderately resistant BRSGO (Chapadões-BRS) and susceptible TMG115 RR (TMG) Glycine max genotypes. Differential expression analysis revealed two stages of response to RLN infection and a set of differentially expressed genes (DEGs) in the first stage suggested a pattern-triggered immunity (PTI) in both genotypes. The divergent time-point of DEGs between genotypes was observed four days post-infection, which included the activation of mitogen-activated protein kinase (MAPK) and plant-pathogen interaction genes in the BRS, suggesting the occurrence of an effector-triggered immunity response (ETI) in BRS. The co-expression analyses combined with single nucleotide polymorphism (SNP) uncovered a key element, a transcription factor phytochrome-interacting factor (PIF7) that is a potential regulator of moderate resistance to RLN infection. Two genes for resistance-related leucine-rich repeat (LRR) proteins were found as BRS-specific expressed genes. In addition, alternative splicing analysis revealed an intron retention in a myo-inositol oxygenase (MIOX) transcript, a gene related to susceptibility, may cause a loss of function in BRS.

Immune Modulation Ability of Hepcidin from Teleost Fish.

Antimicrobial peptides (AMP) play an essential role in the innate immune system, modulating the defense response. In a previous study, we demonstrated the antimicrobial activity of synthetic hepcidin (hep20) from rainbow trout (Oncorhynchus mykiss), and its protective effect in European sea bass (Dicentrarchus labrax) challenged with Vibrio anguillarum. Additionally, we described the uptake and distribution of hep20 in different tissues and leukocyte cells. Interestingly, various AMPs characterized in high vertebrates, called host defense peptides (HDPs), also possess immunomodulation activity. For that reason, the present study explores the immunomodulatory abilities of hep20 through in vitro and in vivo studies. First, a monocyte/macrophage RTS-11 cell line from rainbow trout was used to evaluate hep20 effects on pro- and anti-inflammatory cytokines in fish leukocyte cells. Next, the European sea bass juveniles were used to determine if hep20 can regulate the expression of cytokines in fish immune tissues. The results show that hep20 was uptake inner to RTS-11 cells and was able to induce the expression of IL-10, IL-1ß, and TNFα at transcriptional and protein levels. Then, the European sea bass juveniles were given intraperitoneal injections of the peptide. At 1, 3, 7, 14, and 21 days post-injection (dpi), IL-10, IL -1ß, and TNFα mRNA were quantified in the anterior gut, spleen, and head kidney. The hep20 was able to up-regulate cytokine gene expression in these tissues, mainly in the head kidney. Furthermore, the evaluated cytokines showed a cyclical tendency of higher to lesser expression. Finally, a bioinformatics analysis showed that the structure adopted by hep20 is similar to the γ-core domain described for cysteine-stabilized AMP, defined as immunomodulatory and antimicrobial, which could explain the ability of hep20 to regulate the cytokine expression. This study provides new insights into immunomodulatory function complementary to the previously established antimicrobial activity of hep20, suggesting a role as an HDP in teleost fish. These facts are likely to be associated with molecular functions underpinning the protective effect of fish hepcidin against pathogens.

Antibiofilm and immunomodulatory resorbable nanofibrous filing for dental pulp regenerative procedures.

Multifunctional scaffolds with host defense peptides designed for regenerative endodontics are desirable nanobiotechnological tools for dentistry. Here, different scaffolds were tested for use during the pulp revascularization process, including poly(vinyl alcohol)-PVA hydrogels or resins, collagen hydrogels and poly(vinyl alcohol) PVA/Chitosan (PVA/CS) nanofibers. Based on time to degradation (21 days), nanofibers were chosen to be incorporated with ciprofloxacin and IDR-1002 (each at 50 mg/g). Nanofibers containing ciprofloxacin and IDR-1002 had anti-biofilm activity against Enterococcus faecalis, Staphylococcus aureus and a multispecies oral biofilm, besides anti-inflammatory activities. The in vivo subcutaneous tissue response to tooth fragments filled with nanofibers demonstrated a pulp-like tissue formation, when compared to empty teeth fragments. Thus, we designed a strong antimicrobial, immunomodulatory and regenerative candidate for pulp revascularization and regeneration procedures.

Recent Advances in the Discovery and Function of Antimicrobial Molecules in Platelets.

The conventional function described for platelets is maintaining vascular integrity. Nevertheless, increasing evidence reveals that platelets can additionally play a crucial role in responding against microorganisms. Activated platelets release molecules with antimicrobial activity. This ability was first demonstrated in rabbit serum after coagulation and later in rabbit platelets stimulated with thrombin. Currently, multiple discoveries have allowed the identification and characterization of PMPs (platelet microbicidal proteins) and opened the way to identify kinocidins and CHDPs (cationic host defense peptides) in human platelets. These molecules are endowed with microbicidal activity through different mechanisms that broaden the platelet participation in normal and pathologic conditions. Therefore, this review aims to integrate the currently described platelet molecules with antimicrobial properties by summarizing the pathways towards their identification, characterization, and functional evaluation that have promoted new avenues for studying platelets based on kinocidins and CHDPs secretion.

Big defensin from the scallop Argopecten purpuratus ApBD1 is an antimicrobial peptide which entraps bacteria through nanonets formation.

Big defensins is a large family of antimicrobial peptides found in restricted groups of invertebrates, in particular mollusks where they have highly diversified. Big defensins are composed of a highly hydrophobic N-terminal region and a C-terminal region containing six cysteine residues whose arrangement is identical to that of vertebrate ß-defensins. They have been shown to be active against both Gram-positive and Gram-negative bacteria and fungi. Antimicrobial aggregates called nanonets entrapping and killing bacteria have been recently described for the hydrophobic N-terminal region of the Cg-BigDef1 from the oyster Crassostrea gigas. To determine whether nanonets formation is a conserved trait of mollusk big defensins, we assessed the potential entrapping of bacteria through nanonets of the big defensin from the scallop Argopecten purpuratus, ApBD1. Recombinant ApBD1 was produced with a thrombin-cleavable N-terminal His6 tag, followed by the mature peptide carrying a mutation of the last cysteine residue of the C-terminal region by and arginine, named rApBD1(C87R). This mutation did not apparently affect the three-dimensional structure and the biological properties of rApBD1(C87R), as evidenced by in silico modeling and in vitro antimicrobial assays. Strong immune staining of rApBD1(C87R) in numerous areas surrounding bacteria was observed by confocal microscopy, suggesting that rApBD1(C87R) entraps bacteria in peptide aggregates similar to those reported to the oyster big defensin. This study suggests the conservation of bactericidal activity and nanonet formation across big defensins from bivalve mollusks.

Are Host Defense Peptides and Their Derivatives Ready to be Part of the Treatment of the Next Coronavirus Pandemic?

The term host defense peptides arose at the beginning to refer to those peptides that are part of the host's immunity. Because of their broad antimicrobial capacity and immunomodulatory activity, nowadays, they emerge as a hope to combat resistant multi-drug microorganisms and emerging viruses, such as the case of coronaviruses. Since the beginning of this century, coronaviruses have been part of different outbreaks and a pandemic, and they will be surely part of the next pandemics, this review analyses whether these peptides and their derivatives are ready to be part of the treatment of the next coronavirus pandemic.

Host defense peptides as immunomodulators: The other side of the coin.

Host defense peptides (HDPs) exhibit a broad range of antimicrobial and immunomodulatory activities. In this sense, both functions are like different sides of the same coin. The direct antimicrobial side was discovered first, and widely studied for the development of anti-infective therapies. In contrast, the immunomodulatory side was recognized later and in the last 20 years the interest in this field has been continuously growing. Different to their antimicrobial activities, the immunomodulatory activities of host defense peptides are more effective in vivo. They offer a great opportunity for new therapeutic applications in the fields of anti-infective therapy, chronic inflammatory diseases treatment, novel vaccine adjuvants development and anticancer immunotherapy. These immune related functions of HDPs includes chemoattraction of leukocytes, modulation of inflammation, enhancement of antigen presentation and polarization of adaptive immune responses. Our attempt with this review is to make a careful evaluation of different aspects of the less explored, but attractive immunomodulatory side of the HDP functional coin.

Multifaceted MRGPRX2: New insight into the role of mast cells in health and disease.

Cutaneous mast cells (MCs) express Mas-related G protein-coupled receptor-X2 (MRGPRX2; mouse ortholog MrgprB2), which is activated by an ever-increasing number of cationic ligands. Antimicrobial host defense peptides (HDPs) generated by keratinocytes contribute to host defense likely by 2 mechanisms, one involving direct killing of microbes and the other via MC activation through MRGPRX2. However, its inappropriate activation may cause pseudoallergy and likely contribute to the pathogenesis of rosacea, atopic dermatitis, allergic contact dermatitis, urticaria, and mastocytosis. Gain- and loss-of-function missense single nucleotide polymorphisms in MRGPRX2 have been identified. The ability of certain ligands to serve as balanced or G protein-biased agonists has been defined. Small-molecule HDP mimetics that display both direct antimicrobial activity and activate MCs via MRGPRX2 have been developed. In addition, antibodies and reagents that modulate MRGPRX2 expression and signaling have been generated. In this article, we provide a comprehensive update on MrgprB2 and MRGPRX2 biology. We propose that harnessing MRGPRX2's host defense function by small-molecule HDP mimetics may provide a novel approach for the treatment of antibiotic-resistant cutaneous infections. In contrast, MRGPRX2-specific antibodies and inhibitors could be used for the modulation of allergic and inflammatory diseases that are mediated via this receptor.

Retinoic acid induces antimicrobial peptides and cytokines leading to Mycobacterium tuberculosis elimination in airway epithelial cells.

Tuberculosis (TB) is the leading cause of death by a single infectious agent, Mycobacterium tuberculosis (Mtb). Alveolar macrophages and respiratory epithelial cells are the first cells exposed to Mtb during the primary infection, once these cells are activated, secrete cytokines and antimicrobial peptides that are associated with the Mtb contention and elimination. Vitamins are micronutrients that function as boosters on the innate immune system, however, is unclear whether they have any protective activity during Mtb infection. Thus, we investigated the role of vitamin A (retinoic acid), vitamin C (ascorbic acid), vitamin D (calcitriol), and vitamin E (alfa-tocopherol) as inductors of molecules related to mycobacterial infection in macrophages and epithelial cells. Our results showed that retinoic acid promotes the expression of pro- and anti-inflammatory molecules such as Thymic stromal lymphopoietin (TSLP), ß-defensin-2, IL-1ß, CCL20, ß-defensin-3, Cathelicidin LL-37, TGF-ß, and RNase 7, whereas calcitriol, ascorbic acid, and α-tocopherol lead to an anti-inflammatory response. Treatment of Mtb-infected epithelial cells and macrophage-like cells with the vitamins showed a differential response, where calcitriol reduced Mtb in macrophages, while retinoic acid reduced infection in epithelial cells. Thereby, we propose that a combination of calcitriol and retinoic acid supplementation can drive the immune response, and promotes the Mtb elimination by increasing the expression of antimicrobial peptides and cytokines, while simultaneously modulating inflammation.

Platelets immune response against Mycobacterium tuberculosis infection.

Tuberculosis (TB) is the first cause of death by a single infectious agent. Previous reports have highlighted the presence of platelets within Tb granulomas, albeit the immune-associated platelet response to Mycobacterium tuberculosis (Mtb) has not been deeply studied. Our results showed that platelets are recruited into the granuloma in the late stages of tuberculosis. Furthermore, electron-microscopy studies showed that platelets can internalize Mtb and produce host defense peptides (HDPs), such as RNase 7, HBD2 and hPF-4 that bind to the internalized Mtb. Mtb-infected platelets exhibited higher transcription and secretion of IL-1ß and TNF-α, whereas IL-10 and IL-6 protein levels decreased. These results suggest that platelets participate in the immune response against Mtb through HDPs and cytokines production.

The use of host defense peptides in root canal therapy in rats.

OBJECTIVES: In order to evaluate host defense peptides (HDPs) HHC-10 and synoeca-MP activity in in vitro osteoclastogenesis process and in vivo induced apical periodontitis, testing the effect of molecules in the inflammatory response and in apical periodontitis size/volume after root canal treatment. MATERIALS AND METHODS: In vitro osteoclastogenesis was assessed on bone marrow cell cultures extracted from mice, while in vivo endodontic treatment involved rats treated with Ca(OH)2 or HDPs. In vitro osteoclasts were subjected to TRAP staining, and in vivo samples were evaluated by radiographic and tomographic exams, as well as histologic analysis. RESULTS: None of the substances downregulated the in vitro osteoclastogenesis. Nevertheless, all treatments affected the average of apical periodontitis size in rats, although only teeth treated with HDPs demonstrated lower levels of the inflammatory process. These results demonstrated the in vivo potential of HDPs. Radiographic analysis suggested that HHC-10 and synoeca-MP-treated animals presented a similar lesion size than Ca(OH)2-treated animals after 7-day of endodontic treatment. However, tomography analysis demonstrated smaller lesion volume in synoeca-MP-treated animals than HHC-10 and Ca(OH)2-treated animals, after 7 days. CONCLUSIONS: These molecules demonstrated an auxiliary effect in endodontic treatment that might be related to its immunomodulatory ability, broad-spectrum antimicrobial activity, and possible induction of tissue repair at low concentrations. These results can encourage further investigations on the specific mechanisms of action in animal models to clarify the commercial applicability of these biomolecules for endodontic treatment. CLINICAL SIGNIFICANCE: HDPs have the potential to be adjuvant substances in endodontic therapy due to its potential to reduce inflammation in apical periodontitis.

Host defense peptide IDR-1002 associated with ciprofloxacin as a new antimicrobial and immunomodulatory strategy for dental pulp revascularization therapy.

Regenerative therapies such as dental pulpal revascularization appear as an option for traumatized immature permanent teeth. However, the triple antibiotic paste - TAP (metronidazole, minocycline, and ciprofloxacin), used for these therapies, can generate cytotoxicity and dentin discoloration. In contrast, host defense peptides (HDPs) are promising antimicrobial and immunomodulatory biomolecules for dentistry. This study aimed to evaluate in vitro the antimicrobial activity (against Staphylococcus aureus and Enterococcus faecalis) and the immunomodulatory potential (by the evaluation of IL-1α, IL-6, IL-12, IL-10, TNF-α and NO, in RAW 264.7 macrophages and IL-6, TGF-ß and NO, in L929 fibroblast) of synthetic peptides (DJK-6, IDR-1018, and IDR-1002), compared to TAP in an in vitro infection model containing heat-killed antigens from E. faecalis and S. aureus. Furthermore, the synergistic potential of ciprofloxacin and IDR-1002 was evaluated by checkerboard. Ciprofloxacin was the best antimicrobial of TAP, besides acting in synergism with IDR-1002. TAP was pro-inflammatory (p < 0.05), while the association of ciprofloxacin and IDR-1002 presented an anti-inflammatory profile mainly in the presence of both heat-killed antigens (p < 0.05). Based on these results, ciprofloxacin associated with IDR-1002 may demonstrate an efficient antimicrobial and immunomodulatory action in this in vitro model. Further in vivo studies may determine the real potential of this combination.

Host Defense Effectors Expressed by Hemocytes Shape the Bacterial Microbiota From the Scallop Hemolymph.

The interaction between host immune response and the associated microbiota has recently become a fundamental aspect of vertebrate and invertebrate animal health. This interaction allows the specific association of microbial communities, which participate in a variety of processes in the host including protection against pathogens. Marine aquatic invertebrates such as scallops are also colonized by diverse microbial communities. Scallops remain healthy most of the time, and in general, only a few species are fatally affected on adult stage by viral and bacterial pathogens. Still, high mortalities at larval stages are widely reported and they are associated with pathogenic Vibrio. Thus, to give new insights into the interaction between scallop immune response and its associated microbiota, we assessed the involvement of two host antimicrobial effectors in shaping the abundances of bacterial communities present in the scallop Argopecten purpuratus hemolymph. To do this, we first characterized the microbiota composition in the hemolymph from non-stimulated scallops, finding both common and distinct bacterial communities dominated by the Proteobacteria, Spirochaetes and Bacteroidetes phyla. Next, we identified dynamic shifts of certain bacterial communities in the scallop hemolymph along immune response progression, where host antimicrobial effectors were expressed at basal level and early induced after a bacterial challenge. Finally, the transcript silencing of the antimicrobial peptide big defensin ApBD1 and the bactericidal/permeability-increasing protein ApLBP/BPI1 by RNA interference led to an imbalance of target bacterial groups from scallop hemolymph. Specifically, a significant increase in the class Gammaproteobacteria and the proliferation of Vibrio spp. was observed in scallops silenced for each antimicrobial. Overall, our results strongly suggest that scallop antimicrobial peptides and proteins are implicated in the maintenance of microbial homeostasis and are key molecules in orchestrating host-microbiota interactions. This new evidence depicts the delicate balance that exists between the immune response of A. purpuratus and the hemolymph microbiota.