Frank Blecha

Cathelicidins: microbicidal activity, mechanisms of action, and roles in innate immunity.

Antimicrobial peptides are important host-defense molecules of innate immunity. Cathelicidins are a diverse family of potent, rapidly acting and broadly effective antimicrobial peptides, which are produced by a variety of cells. This review examines the classification, antimicrobial spectrum, mechanism of action, and regulation of cathelicidins.

Antimicrobial peptides and bacteriocins: alternatives to traditional antibiotics.

Abstract Antimicrobial peptides (AMPs) are ubiquitous, gene-encoded natural antibiotics that have gained recent attention in the search for new antimicrobials to combat infectious disease. In multicellular organisms, AMPs, such as defensins and cathelicidins, provide a coordinated protective response against infection and are a principal component of innate immunity in vertebrates. In unicellular organisms, AMPs, such as bacteriocins, function to suppress competitor species. Because many AMPs kill bacteria by disruption of membrane integrity and are thus thought to be less likely to induce resistance, AMPs are being extensively evaluated as novel antimicrobial drugs. This review summarizes and discusses the antibiotic properties of AMPs highlighting their potential as alternatives to conventional antibiotics.

CD14 and other recognition molecules for lipopolysaccharide: A review

Lipopolysaccharide (LPS) or endotoxin elicits a broad, non-specific cascade of events in vivo, resulting in secretion of a variety of potent mediators and cytokines produced primarily by activated macrophages and monocytes. The overproduction of these effector molecules, such as interleukin-1 and tumor necrosis factor-alpha, contributes to the pathophysiology of endotoxic shock. Cellular recognition of LPS involves several different molecules, including cluster of differentiation antigen CD14. A thorough understanding of the interaction of LPS with cells of the immune system is necessary before effective preventative or therapeutic measures can be designed to limit the host response to endotoxin. This review discusses the role of CD14 and other LPS-recognition molecules in LPS-mediated macrophage activation.

Chemoattractant properties of PR-39, a neutrophil antibacterial peptide.

The proline‐arginine (PR) ‐rich antibacterial peptide, PR‐39, kills bacteria by a non‐pore‐forming mechanism. Because this neutrophil peptide possesses several distinct functional properties and because other antimicrobial peptides are chemoattractants, we sought to determine whether PR‐39 was a chemoattractant for porcine leukocytes. The peptide was synthesized by the solid‐phase method using t‐Boc chemistry and purified by reversed‐phase high‐performance liquid chromatography. Leukocyte migration was assessed with the use of a 48‐well microchemotaxis chamber. PR‐39 induced the directed migration of neutrophils. The peak chemotaxis response occurred at 0.5–2 μM, which was slightly lower than the minimal inhibitory and bactericidal concentrations for PR‐39. However, the peptide was not a chemoattractant for mononuclear cells. Truncation of PR‐39 suggested that the neutrophil chemoattractant domain may be contained within the first 26 amino acid residues. Intracellular Ca2+ fluxes in response to PR‐39 were monitored by flow cytometry and showed a transient increase that peaked at approximately 40 s and approached basal values by 4 min. However, in a Ca2+‐free environment, the PR‐39‐induced Ca2+ increase was abrogated. Furthermore, PR‐39 did not induce neutrophil chemotaxis in the absence of extracellular Ca2+, and pertussis toxin inhibited both neutrophil chemotaxis and Ca2+ mobilization. Taken together, these data suggest that PR‐39 is a Ca2+‐dependent chemoattractant of neutrophils. The finding of a neutrophil antibacterial peptide that is also a neutrophil chemoattractant is intriguing and may indicate an important role for PR‐39 in inflammation. J. Leukoc. Biol. 61: 624–629; 1997.

Porcine host defense peptides: Expanding repertoire and functions

Host defense peptides (HDPs) are a large group of innate immune effectors that are also termed antimicrobial peptides. Because of the rapid progress that has been made in completing several animal genomes, many HDPs have been systemically defined using bioinformatic analysis and partially characterized using reverse genomic approaches. In pigs, about 30 HDPs have been identified and partially characterized relative to structure and function. Antimicrobial activity of porcine HDPs has been extensively evaluated against a broad spectrum of microorganisms in vitro and evaluated for their protective role in vivo. Increasing evidence indicates that HDPs are functionally differentiated during posttranslational and postsecretory processing, and that the structural units for antimicrobial and immunoregulatory functions are separate. These findings suggest promising new avenues for therapeutic drug design based on HDPs, including porcine HDPs. This review summarizes and discusses advances in porcine HDPs research during the last decade with an emphasis on the rapidly expanding profiles and biological functions.

Molecular cloning and tissue expression of porcine β-defensin-1

Beta‐defensins constitute an emerging family of cysteine‐rich antimicrobial peptides, which are particularly prominent at mucosal epithelial sites in mammals. Here we report the identification of a novel β‐defensin from porcine tissues, porcine β‐defensin‐1 (pBD‐1). The cDNA sequence of pBD‐1 encoded a 64 amino acid prepro‐peptide, which contained the β‐defensin consensus sequence of six invariantly spaced cysteine residues. Northern blot analysis showed that pBD‐1 was expressed abundantly in tongue epithelia and that the expression was regulated developmentally. Using RT‐PCR, pBD‐1 mRNA was detected throughout the respiratory and digestive tracts and also in thymus, spleen, lymph node, brain, liver, kidney, urinary bladder, testis, skin, heart, muscle, bone marrow, peripheral blood neutrophils, alveolar macrophages, and umbilical cord. The wide expression of pBD‐1 suggests that this endogenous peptide antibiotic may contribute to both mucosal and systemic host defenses in pigs, which may have implications for the use of porcine tissues and organs in xenotransplantation.

Regulation of cathelicidin gene expression: induction by lipopolysaccharide, interleukin-6, retinoic acid, and Salmonella enterica serovar typhimurium infection.

ABSTRACT Cathelicidins are a family of antimicrobial peptides prominent in the host defense mechanisms of several mammalian species. In addition to their antimicrobial activities, these peptides have been implicated in wound healing, angiogenesis, and other innate immune mechanisms. To investigate the regulatory mechanisms of cathelicidin gene expression, we conducted in vitro experiments evaluating the bone marrow cell expression of two porcine cathelicidins, PR-39 and protegrin, and cloned and evaluated the promoter sequence of PR-39. In addition, we evaluated in vivo kinetics of cathelicidin gene expression in pigs during an infection with Salmonella entericaserovar Typhimurium. Lipopolysaccharide (LPS) increased PR-39 and protegrin mRNA expression, which was ameliorated by polymyxin B. Concentrations of PR-39 in supernatants from bone marrow cell cultures were increased 10-fold after LPS stimulation. Similarly, interleukin-6 (IL-6) and all-trans retinoic acid (RA) markedly induced cathelicidin gene expression. To verify the transcriptional activation of the PR-39 gene by these agents, we made a PR-39 promoter-luciferase construct containing the full-length PR-39 promoter driving luciferase gene expression and transiently transfected PK-15 epithelial cells. RA and IL-6 increased luciferase activity in PK-15 cells transfected with the PR-39 promoter-luciferase reporter. Similarly,Salmonella-challenged pigs showed increased expression of PR-39 and protegrin mRNA in bone marrow cells at 6 and 24 h postchallenge. Taken together, these findings show that bacterial products (LPS), IL-6, RA, and Salmonella infection enhance the expression of the cathelicidins, PR-39 and protegrin, in bone marrow progenitor cells, and we suggest that extrinsic modulation of this innate host defense mechanism may be possible.

Structural and functional annotation of the porcine immunome

BackgroundThe domestic pig is known as an excellent model for human immunology and the two species share many pathogens. Susceptibility to infectious disease is one of the major constraints on swine performance, yet the structure and function of genes comprising the pig immunome are not well-characterized. The completion of the pig genome provides the opportunity to annotate the pig immunome, and compare and contrast pig and human immune systems.ResultsThe Immune Response Annotation Group (IRAG) used computational curation and manual annotation of the swine genome assembly 10.2 (Sscrofa10.2) to refine the currently available automated annotation of 1,369 immunity-related genes through sequence-based comparison to genes in other species. Within these genes, we annotated 3,472 transcripts. Annotation provided evidence for gene expansions in several immune response families, and identified artiodactyl-specific expansions in the cathelicidin and type 1 Interferon families. We found gene duplications for 18 genes, including 13 immune response genes and five non-immune response genes discovered in the annotation process. Manual annotation provided evidence for many new alternative splice variants and 8 gene duplications. Over 1,100 transcripts without porcine sequence evidence were detected using cross-species annotation. We used a functional approach to discover and accurately annotate porcine immune response genes. A co-expression clustering analysis of transcriptomic data from selected experimental infections or immune stimulations of blood, macrophages or lymph nodes identified a large cluster of genes that exhibited a correlated positive response upon infection across multiple pathogens or immune stimuli. Interestingly, this gene cluster (cluster 4) is enriched for known general human immune response genes, yet contains many un-annotated porcine genes. A phylogenetic analysis of the encoded proteins of cluster 4 genes showed that 15% exhibited an accelerated evolution as compared to 4.1% across the entire genome.ConclusionsThis extensive annotation dramatically extends the genome-based knowledge of the molecular genetics and structure of a major portion of the porcine immunome. Our complementary functional approach using co-expression during immune response has provided new putative immune response annotation for over 500 porcine genes. Our phylogenetic analysis of this core immunome cluster confirms rapid evolutionary change in this set of genes, and that, as in other species, such genes are important components of the pig’s adaptation to pathogen challenge over evolutionary time. These comprehensive and integrated analyses increase the value of the porcine genome sequence and provide important tools for global analyses and data-mining of the porcine immune response.

Porcine alveolar and pulmonary intravascular macrophages: comparison of immune functions.

Although a substantial amount of information is available on pulmonary alveolar macrophages (PAMs), little is known about pulmonary intravascular macrophages (PIMs), a separate population of lung macrophages found apposed to the endothelium of pulmonary capillaries. We compared these two populations of lung immunocytes to determine their relative immunological activity. Our results suggest that PAMs are more phagocytic than PIMs; however, PIMs may be more efficient at lysing ingested bacteria than PAMs. Although similar in antibody‐dependent cellular cytotoxicity, PIMs are more spontaneously cytolytic than PAMs. Depending upon the effector:target cell ratio studied, the tumoricidal activity of PIMs was less than or equal to that of PAMs. Both cell populations produced the cytokines interleukin‐1 and tumor necrosis factor‐α at similar concentrations. These data suggest that PIMs are immunologically active, although the degree of activity may differ between PIMs and PAMs.

Identification of a proline-arginine-rich antibacterial peptide from neutrophils that is analogous to PR-39, an antibacterial peptide from the small intestine.

Neutrophil nonoxidative defense mechanisms include several low molecular weight antimicrobial peptides. We have isolated a proline‐arginine‐rich antibacterial peptide from porcine neutrophils. This cationic, low molecular weight peptide has a very high degree of identity (97%) to a bactericidal peptide, PR‐39, that has been found in the porcine small intestine. Isolation of the same antimicrobial peptide from both neutrophils and cells of the small intestine in the same species is unique and suggests an important role for this protein in innate immune defenses. J. Leukoc. Biol. 56: 807–811; 1994.