Research Note: Intestinal avian defensin 2 and robustness of chicks.

Poultry production is an important agricultural sector for human food worldwide. Chicks after hatch often face health problems leading to economic losses that are deleterious for breeders. Avian defensin 2 (AvBD2) is a prominent host defense peptide of the intestinal mucosa of cecum and is involved in the resistance of poultry to bacterial pathogens. This peptide could thus represent an innate immunity marker of robustness of birds. To test this hypothesis by comparing fast-growing and slow-growing lines in different conditions of breeding, the chick's cecal AvBD2 content was analyzed according to animal quality and immunity indicators. Chick's cecal tissue sections labeled by immunohistochemistry with newly developed specific antibodies revealed the localization of AvBD2 in the mucosa with high individual variability, without showing differences attributable to quality indicators, but interestingly showing inverse correlation with seric IgM levels in the fast-growing line. The availability of our anti-AvBD2 antibodies to the scientific community opens perspectives to identify the cellular sources of this defensin in the cecal mucosa and to investigate the organization and function of innate immune arsenal of birds.

Evolutionary diversification of defensins and cathelicidins in birds and primates.

Divergent evolution for more than 310 million years has resulted in an avian immune system that is complex and more compact than that of primates, sharing much of its structure and functions. Not surprisingly, well conserved ancient host defense molecules, such as defensins and cathelicidins, have diversified over time. In this review, we describe how evolution influenced the host defense peptides repertoire, its distribution, and the relationship between structure and biological functions. Marked features of primate and avian HDPs are linked to species-specific characteristics, biological requirements, and environmental challenge.

Upregulation of gut cathepsin L during Eimeria tenella infection.

Coccidiosis is a disease caused by Eimeria, which represents the first parasitic disease in poultry farming. Among them, E. tenella is a virulent species which specifically colonizes the caecum. The inflammatory response to infection is associated to numerous host proteases including cysteine cathepsins that can be deleterious for tissue and innate immunity integrity. Here, germ-free and conventional chickens were used as models to find out whether the microbiota could modify the intestinal expression of host cysteine cathepsins during coccidiosis. The basal caecal peptidase activity primarily relies on host proteases rather than proteases from the commensal flora. While mRNA levels of E. tenella cathepsins B and L remained unchanged in germ-free and conventional broilers, an overall increase in endopeptidase activity of cysteine cathepsins was found in E. tenella-infected caeca in both experimental models (P < 0.005). A significant decrease in avian cystatin C transcription was also observed in infected conventional, but not in infected germ-free broilers. Despite an unchanged mRNA level of avian cathepsin L (CatL), its protein expression raised following infection, in parallel with an increased transcription of antimicrobial ß-defensins (AvBD1, AvBD2, AvBD4, AvBD6, and AvBD7). Taken together, data support that host CatL is post-translationally upregulated during E. tenella infection, and thus may be involved in the alteration of the gut proteolytic balance. Furthermore, CatL may participate to inflammation occurring during coccidiosis through its known ability to proteolytically inactivates up-regulated avian ß-defensins that are key molecules of innate immunity.

Systemic Administration of Avian Defensin 7: Distribution, Cellular Target, and Antibacterial Potential in Mice.

Defensins are natural antimicrobial peptides. The avian beta-defensin AvBD7 isolated from the chicken bone marrow possess broad antibacterial spectrum and strong resistance to proteolysis. However, its ability to fight systemic infections of major concern for public health, such as salmonellosis, is unknown. As a first approach, fluorescence labeling of AvBD7 allowed to track its systemic distribution after intraperitoneal injection in mice using whole body live imaging. It was associated to peritoneal cells and to deeper organs such as the liver. In the next step, the use of labeled AvBD7 allowed to observe its interaction with murine macrophages in culture. After incubation, it was able to penetrate inside the cells through an endocytosis-like mechanism. Furthermore, natural AvBD7 contributed to the control of intracellular multiplication of a multidrug resistant Salmonella strain, after incubation with infected macrophages. Finally, administration in a model of systemic lethal Salmonella infection in mice led to significant improvement of mouse survival, consistently with significant reduction of the liver bacterial load. In conclusion, the results reveal a hitherto unknown intracellular antibacterial effect of AvBD7 in Salmonella target cells and support AvBD7 as a candidate of interest for the treatment of infectious diseases caused by multidrug-resistant pathogenic Enterobacteriaceae.

Unveiling the participation of avian kinin ornithokinin and its receptors in the chicken inflammatory response.

Vasoactive peptides are key early mediators of inflammation released through activation of different enzymatic systems. The mammalian kinin-kallikrein (K-KLK) system produces bradykinin (BK) through proteolytic cleavage of a kininogen precursor by enzymes named kallikreins. BK acts through specific ubiquitous G-protein coupled receptors (B1R and B2R) to participate in physiological processes and inflammatory responses, such as activation of mononuclear phagocytes. In chickens, the BK-like nonapeptide ornithokinin (OK) has been shown to promote intracellular calcium increase in embryonic fibroblasts and to be vasodilatory in vivo. Also, one of its receptors (B2R) was already cloned. However, the participation of chicken K-KLK system components in the inflammatory response remains unknown and was therefore investigated. We first showed that B1R, B2R and kininogen 1 (KNG1) are expressed in unstimulated chicken tissues and macrophages. We next showed that chicken B1R and B2R are expressed at transcript and protein levels in chicken macrophages and are upregulated by E. coli LPS or avian pathogenic E. coli (APEC) infection. Interestingly, exogenous OK induced internalization and degradation of OK receptors protein, notably B2R. Also, OK induced intracellular calcium increase and potentiated zymosan-induced ROS production and Dextran-FITC endocytosis by chicken macrophages. Exogenous OK itself did not promote APEC killing and had no pro-inflammatory effect. However, when combined with LPS or APEC, OK upregulated cytokine/chemokine gene expression and NO production by chicken macrophages. This effect was not blocked by canonical non-peptide B1R or B2R receptor antagonists but was GPCR- and PI3K/Akt-dependent. In vivo, pulmonary colibacillosis led to upregulation of OK receptors expression in chicken lungs and liver. Also, colibacillosis led to significant upregulation of OK precursor KNG1 expression in liver and in cultured hepatocytes (LMH). We therefore provide hitherto unknown information on how OK and its receptors are involved in inflammation and infection in chickens.

Complete Genome Sequences of Two Escherichia coli Phages, vB_EcoM_ ESCO5 and vB_EcoM_ESCO13, Which Are Related to phAPEC8.

We report here the complete genome sequences of two Myoviridae phages that infect various avian-pathogenic Escherichia coli strains and that are closely related to phage phAPEC8.

The Unusual Resistance of Avian Defensin AvBD7 to Proteolytic Enzymes Preserves Its Antibacterial Activity.

Defensins are frontline peptides of mucosal immunity in the animal kingdom, including birds. Their resistance to proteolysis and their ensuing ability to maintain antimicrobial potential remains questionable and was therefore investigated. We have shown by bottom-up mass spectrometry analysis of protein extracts that both avian beta-defensins AvBD2 and AvBD7 were ubiquitously distributed along the chicken gut. Cathepsin B was found by immunoblotting in jejunum, ileum, caecum, and caecal tonsils, while cathepsins K, L, and S were merely identified in caecal tonsils. Hydrolysis product of AvBD2 and AvBD7 incubated with a panel of proteases was analysed by RP-HPLC, mass spectrometry and antimicrobial assays. AvBD2 and AvBD7 were resistant to serine proteases and to cathepsins D and H. Conversely cysteine cathepsins B, K, L, and S degraded AvBD2 and abolished its antibacterial activity. Only cathepsin K cleaved AvBD7 and released Ile4-AvBD7, a N-terminal truncated natural peptidoform of AvBD7 that displayed antibacterial activity. Besides the 3-stranded antiparallel beta-sheet typical of beta-defensins, structural analysis of AvBD7 by two-dimensional NMR spectroscopy highlighted the restricted accessibility of the C-terminus embedded by the N-terminal region and gave a formal evidence of a salt bridge (Asp9-Arg12) that could account for proteolysis resistance. The differential susceptibility of avian defensins to proteolysis opens intriguing questions about a distinctive role in the mucosal immunity against pathogen invasion.

Antimicrobial Peptide LL-37 Is Both a Substrate of Cathepsins S and K and a Selective Inhibitor of Cathepsin L.

Lung cysteine cathepsins B, K, L, and S contribute to physiological and pathological processes including degradation of antimicrobial peptides/proteins (AMPs) such as surfactant protein SP-A, lactoferrin, secretory leukocyte peptidase inhibitor, and beta-defensins-2 and -3. Substantial amounts of uncleaved LL-37, a 37-mer cationic AMP, were observed in the sputum of patients with cystic fibrosis (CF). Nevertheless LL-37 was degraded after prolonged incubation in CF sputum, and the hydrolysis was blocked by E-64, a selective inhibitor of cysteine proteases. Cathepsins K and S, expressed in human alveolar macrophages, thoroughly hydrolyzed LL-37 in vitro, whereas it competitively inhibited cathepsin L (Ki = 150 nM). Cleavage of LL-37 by cathepsins S and K impaired its antimicrobial activity against Pseudomonas aeruginosa and Staphylococcus aureus, in a time- and concentration-dependent manner. The exchange of residues 67 and 205 in the S2 pockets of cathepsins L (Leu67Tyr/Ala205Leu) and K (Tyr67Leu/Leu205Ala) switched the specificity of these mutants toward LL-37. Molecular modeling suggested that LL-37 interacted with the active site of cathepsin L in both forward (i.e., substrate-like) and reverse orientations with similar binding energies. Our data support the hypothesis that cysteine cathepsins modulate the innate immunity response by degrading distinct and representative members of the AMP family.

Protection patterns in duck and chicken after homo- or hetero-subtypic reinfections with H5 and H7 low pathogenicity avian influenza viruses: a comparative study.

Avian influenza viruses are circulating continuously in ducks, inducing a mostly asymptomatic infection, while chickens are accidental hosts highly susceptible to respiratory disease. This discrepancy might be due to a different host response to the virus between these two bird species and in particular to a different susceptibility to reinfection. In an attempt to address this question, we analyzed, in ducks and in chickens, the viral load in infected tissues and the humoral immune response after experimental primary and secondary challenge infections with either homologous or heterologous low pathogenicity avian influenza viruses (LPAIV). Following homologous reinfection, ducks were only partially protected against viral shedding in the lower intestine in conjunction with a moderate antibody response, whereas chickens were totally protected against viral shedding in the upper respiratory airways and developed a stronger antibody response. On the contrary, heterologous reinfection was not followed by a reduced viral excretion in the upper airways of chickens, while ducks were still partially protected from intestinal excretion of the virus, with no correlation to the antibody response. Our comparative study provides a comprehensive demonstration of the variation of viral tropism and control of the host humoral response to LPAIV between two different bird species with different degrees of susceptibility to avian influenza.

Susceptibility to Salmonella carrier-state: a possible Th2 response in susceptible chicks.

Infection of chicken with Salmonella may lead to a carrier-state characterized by the persistence of bacteria in the ceca for a long period of time and result in their excretion in feces. This excretion is the source of contamination of their congeners and food. During infection, enterocytes are the primary target cells for Salmonella, the producers of soluble factors which launch immune response and cells which are reciprocally responsive to surrounding immune cells. This study used microarrays to compare the gene expression profile during carrier-state of enterocytes purified from infected and control chicks which are either resistant or susceptible to Salmonella Enteritidis carrier-state. In total, we identified 271 genes significantly differentially expressed with an absolute fold change greater than 1.5. A global analysis determined interaction networks between differentially regulated genes. Using an a priori approach, our analyses focused on differentially expressed genes which were transcriptionally linked to cytokines playing a major role in the fate of the immune response. The expression of genes transcriptionally linked to type I interferon and TGF-ß was down-regulated in infected chicks from both lines. Gene expression linked to the Th1 axis suggests the latter is inhibited in both lines. Finally, the expression of genes linked to IL-4, IL-5 and IL-13 indicates that susceptibility to carrier-state could be associated with a Th2 bias. Overall, these results highlight that the response to Salmonella during the acute phase and carrier-state is different and that enterocytes play a central role in this response.

Initial insights into structure-activity relationships of avian defensins.

Numerous ß-defensins have been identified in birds, and the potential use of these peptides as alternatives to antibiotics has been proposed, in particular to fight antibiotic-resistant and zoonotic bacterial species. Little is known about the mechanism of antibacterial activity of avian ß-defensins, and this study was carried out to obtain initial insights into the involvement of structural features or specific residues in the antimicrobial activity of chicken AvBD2. Chicken AvBD2 and its enantiomeric counterpart were chemically synthesized. Peptide elongation and oxidative folding were both optimized. The similar antimicrobial activity measured for both L- and D-proteins clearly indicates that there is no chiral partner. Therefore, the bacterial membrane is in all likelihood the primary target. Moreover, this work indicates that the three-dimensional fold is required for an optimal antimicrobial activity, in particular for gram-positive bacterial strains. The three-dimensional NMR structure of chicken AvBD2 defensin displays the structural three-stranded antiparallel ß-sheet characteristic of ß-defensins. The surface of the molecule does not display any amphipathic character. In light of this new structure and of the king penguin AvBD103b defensin structure, the consensus sequence of the avian ß-defensin family was analyzed. Well conserved residues were highlighted, and the potential strategic role of the lysine 31 residue of AvBD2 was emphasized. The synthetic AvBD2-K31A variant displayed substantial N-terminal structural modifications and a dramatic decrease in activity. Taken together, these results demonstrate the structural as well as the functional role of the critical lysine 31 residue in antimicrobial activity.

Purification and characterization of avian beta-defensin 11, an antimicrobial peptide of the hen egg.

Natural antimicrobial peptides are present in different compartments (eggshell, egg white, and vitelline membranes) of the hen egg and are expected to be involved in the protection of the embryo during its development and to contribute to the production of pathogen-free eggs. In the present study, we used vitelline membranes from hen (Gallus gallus) eggs as a source of avian ß-defensin 11 (AvBD11). A purification scheme using affinity chromatography and reverse-phase chromatography was developed. Purified AvBD11 was analyzed by a combination of mass spectrometry approaches to characterize its primary sequence and structure. A monoisotopic molecular species at [M + H](+) of 9,271.56 Da was obtained, and its N- and C-terminal sequences were determined. We also examined posttranslational modifications and identified the presence of 6 internal disulfide bonds. AvBD11 was found to exhibit antimicrobial activity toward both Gram-positive and Gram-negative bacteria.

Primary structure and antibacterial activity of chicken bone marrow-derived beta-defensins.

Three biologically active beta-defensins were purified by chromatography from chicken bone marrow extract: avian beta-defensin 1 (AvBD1), AvBD2, and the newly isolated beta-defensin AvBD7. Mass spectrometry analyses showed that bone marrow-derived AvBD1, -2, and -7 peptides were present as mature peptides and revealed posttranslational modifications for AvBD1 and AvBD7 in comparison to their in silico-predicted amino acid sequences. Tandem mass spectrometry analysis using the nanoelectrospray-quadrupole time of flight method showed N-terminal glutaminyl cyclization of mature AvBD7 and C-terminal amidation of mature AvBD1 peptide, while posttranslational modifications were absent in bone marrow-derived mature AvBD2 peptide. Furthermore, mass spectrometry analysis performed on intact cells confirmed the presence of these three peptides in mature heterophils. In addition, the antibacterial activities of the three beta-defensins against a large panel of gram-positive and -negative bacteria were assessed. While the three defensins displayed similar antibacterial spectra of activity against gram-positive strains, AvBD1 and AvBD7 exhibited the strongest activity against gram-negative strains in comparison to AvBD2.

Differential modulation of beta-defensin gene expression by Salmonella Enteritidis in intestinal epithelial cells from resistant and susceptible chicken inbred lines.

beta-Defensins are important components of innate immunity in mucosal tissue, a major entry site for several pathogens. These small cationic peptides possess antimicrobial activity against various microorganisms including Salmonella. Two chicken inbred lines, 6 and 15I, diverge phenotypically with respect to levels of Salmonella Enteritidis intestinal carriage and to level of gene expression of two beta-defensins, AvBD1 and AvBD2. The cellular source of these two defensins in the intestinal tissue has not previously been explored. Therefore embryonic intestinal cells were isolated from both chicken lines. Primary intestinal cell cultures expressed epithelial specific markers (villin and E-cadherin) and differentially expressed two beta-defensin genes AvBD1 and AvBD2 according to chicken line. Furthermore, S. Enteritidis interfered with AvBD2 expression only in the cells from the susceptible line 15I. Our embryonic cell culture model demonstrated that intestinal epithelium express beta-defensin antimicrobial peptides that may play a role in immunoprotection against Salmonella Enteritidis.

Salmonella carrier-state in hens: study of host resistance by a gene expression approach.

Salmonellosis is one of the main causes of food-borne poisoning due to the consumption of contaminated poultry products. In the flocks, Salmonella is able to persist in the digestive tract of birds for weeks without triggering any symptom. In order to identify molecules and genes involved in the mechanism of host resistance to intestinal carrier-state, two different inbred lines of laying hens were orally inoculated with Salmonella Enteritidis. Bacterial colonization and host gene expression were measured in the caecum and its sentinel lymphoid tissue, respectively. Significantly increased expression of chemokine, anti-infectious cytokine, bacterial receptor, antimicrobial mediator and particularly, defensin genes was observed in the line carrying a lower level of bacteria in the caecum. These innate immunity molecules were either constitutively or inductively highly expressed in resistant adult birds and thus present candidate genes to play an important role in the host defence against Salmonella colonization.

Salmonella carrier state in chicken: comparison of expression of immune response genes between susceptible and resistant animals.

Asymptomatic Salmonella enterica serovar Enteritidis carrier state in poultry has serious consequences on food safety and public health due to the risks of food poisoning following consumption of contaminated products. An understanding the mechanisms of persistence of Salmonella in the digestive tract of chicken can be achieved by a better knowledge of the defects in the control of infection in susceptible versus resistant animals. The gene expression of innate immune response factors including anti-microbial molecules, inflammatory and anti-infectious cytokines was studied in the caecal lymphoid tissue associated with the carrier state. Expression levels of these genes were assessed by real-time PCR and were compared in two inbred lines of chickens differing in resistance to the carrier state following oral inoculation of S. enterica serovar Enteritidis at 1 week of age. No correlation was observed between resistance/susceptibility to caecal carrier state and level of interleukin (IL)-1beta, IL-8, IL-18, inducible NO synthase (iNOS) and natural resistance associated macrophage protein 1 (NRAMP1). A high baseline level of defensin gene expression was recorded in young animals from the susceptible line. In contrast, a significantly low expression of interferon-gamma (IFN-gamma) gene was observed in these susceptible infected animals in comparison to resistant ones and healthy counterparts. IFN-gamma expression level represents a valuable indication of immunodeficiency associated with persistence of Salmonella in the chicken digestive tract, and IFN-gamma thus represents a factor to consider in the development of prophylactic measures for the reduction of Salmonella carrier state.

Expression of interleukin-1 (IL-1) system genes in equine cumulus-oocyte complexes and influence of IL-1beta during in vitro maturation.

A growing body of evidence suggests that the ovary is a site of inflammatory reactions, and thus, ovarian cells could represent sources and targets of the interleukin-1 (IL-1) system. The aim of the present work was to investigate the expression of IL-1alpha, IL-1beta, IL-1ra, IL-1R1, and IL-1R2 genes in equine cumulus cells and oocytes. Moreover, the influence of IL-1beta on in vitro maturation of cumulus-oocytes complexes (COCs) was examined. COCs were collected using ultrasound-guided follicular puncture in vivo. Oocytes and cumulus cells were isolated from preovulatory and subordinate follicles and were analyzed either at collection or after in vitro culture. An ultrasensitive reverse transcription-polymerase chain reaction was used to study the expression of IL-1 system members. In order to study the in vitro effect of IL-1beta on oocyte nuclear maturation and cumulus expansion, immature COCs collected from subordinate follicles were cultured for 30 h in media containing or not containing IL-1beta alone, or in combination with equine luteinizing hormone. Our results indicated that equine oocytes expressed IL-1beta and IL-1R2 genes, and that cumulus cells expressed all IL-1 system members except IL-1alpha. In oocytes, IL-1beta expression significantly decreased during in vitro culture compared with immature and mature oocytes analyzed at collection. Similarly, in cumulus cells, in vitro culture decreased IL-1ra and IL-1R1 expression. IL-1beta was shown to significantly decrease the gonadotropin-induced nuclear maturation of oocytes. In conclusion, we demonstrated for the first time, in mares, the presence of IL-1beta and IL-1R2 transcripts in oocytes and of IL-1beta, IL-1ra, IL-1R1, and IL-1R2 in cumulus cells. The regulatory effect of IL-1beta on COCs during maturation suggests that the IL-1 system could be of crucial importance for ovarian physiology in the mare.