Guolong Zhang

Toll-like receptor–mediated NF-κB activation: a phylogenetically conserved paradigm in innate immunity

Innate immunity is an ancient form of host defense that is shared by almost all multicellular organisms (1, 2). However, it is not a redundant defense mechanism, and recent evidence has shown that innate immunity not only provides a first line of antimicrobial host defense, but also has a profound impact on the establishment of adaptive immune responses (1, 3). Upon infection, microorganisms are first recognized by cells of the host innate immune system, such as phagocytic leukocytes, endothelial and mucosal epithelial cells, and professional antigen-presenting cells. Recognition of pathogens is primarily mediated by a set of germline-encoded molecules on innate immune cells that are referred to as pattern recognition receptors (PRRs) (3). Well characterized PRRs include CD14, β2-integrins (CD11/CD18), C-type lectins, macrophage scavenger receptors, and complement receptors (CR1/CD35, CR2/CD21) (3). These PRRs are expressed as either membrane-bound or soluble proteins that recognize invariant molecular structures called pathogen-associated molecular patterns (PAMPs) that are shared by many pathogens but not expressed by hosts (3). Examples of PAMPs include LPS, bacterial lipoprotein (BLP), peptidoglycan (PGN) lipoteichoic acid (LTA), unmethylated CpG DNA of bacteria, lipoarabinomannan (LAM) of mycobacteria, and mannans of yeast (3). Recognition of PAMPs by PRRs results in the activation of different intracellular signaling cascades that in turn lead to the expression of various effector molecules (3). One group of effector molecules consists of reactive oxygen and nitrogen intermediates and various antimicrobial peptides that have direct microbicidal activity and collectively provide immediate protection for hosts. Another group includes cytokines, chemokines, adhesion molecules, and acute phase proteins that are involved in inflammation and early host defense as well as the development of adaptive immune responses. The third group consists of the costimulatory molecules B7.1 and B7.2, which bind CD28 on T cells and act as the second signal for T-cell activation. Therefore, signaling by the PRRs helps to bridge innate and adaptive immunity and allows the host to cope more efficiently with microbial infection. In keeping with the important role that innate immunity plays in protecting multicellular organisms from infection, components of the innate immune response, including pathogen recognition molecules, signal transduction pathways, and downstream effector molecules, are all evolutionarily conserved and are used by insects, plants, and mammals (2). Recent studies on the recognition of microbial PAMPs have highlighted the critical role of one group of PRRs, the Toll-like receptors (TLRs), in pathogen recognition and host defense. These TLRs are distinguished from other PRRs by their ability to recognize and, more significantly, discriminate between, different classes of pathogens (reviewed in refs. 4, 5). Engagement of TLRs by pathogens leads to the activation of innate immune responses (5), and a major signaling target of the TLRs is activation of the transcription factor NF-κB, a key regulator of immune and inflammatory responses (reviewed in refs. 6–8). Interestingly, TLR-mediated NF-κB activation is also an evolutionarily conserved event that occurs in phylogenetically distinct species ranging from insects to mammals (5, 9, 10). Here, we focus on the role of the conserved TLR/NF-κB signaling pathway in innate immunity, as well as its impact on adaptive immune responses.

A genome-wide screen identifies a single β-defensin gene cluster in the chicken: implications for the origin and evolution of mammalian defensins

BackgroundDefensins comprise a large family of cationic antimicrobial peptides that are characterized by the presence of a conserved cysteine-rich defensin motif. Based on the spacing pattern of cysteines, these defensins are broadly divided into five groups, namely plant, invertebrate, α-, β-, and θ-defensins, with the last three groups being mostly found in mammalian species. However, the evolutionary relationships among these five groups of defensins remain controversial.ResultsFollowing a comprehensive screen, here we report that the chicken genome encodes a total of 13 different β-defensins but with no other groups of defensins being discovered. These chicken β-defensin genes, designated as Gallinacin 1–13, are clustered densely within a 86-Kb distance on the chromosome 3q3.5-q3.7. The deduced peptides vary from 63 to 104 amino acid residues in length sharing the characteristic defensin motif. Based on the tissue expression pattern, 13 β-defensin genes can be divided into two subgroups with Gallinacin 1–7 being predominantly expressed in bone marrow and the respiratory tract and the remaining genes being restricted to liver and the urogenital tract. Comparative analysis of the defensin clusters among chicken, mouse, and human suggested that vertebrate defensins have evolved from a single β-defensin-like gene, which has undergone rapid duplication, diversification, and translocation in various vertebrate lineages during evolution.ConclusionsWe conclude that the chicken genome encodes only β-defensin sequences and that all mammalian defensins are evolved from a common β-defensin-like ancestor. The α-defensins arose from β-defensins by gene duplication, which may have occurred after the divergence of mammals from other vertebrates, and θ-defensins have arisen from α-defensins specific to the primate lineage. Further analysis of these defensins in different vertebrate lineages will shed light on the mechanisms of host defense and evolution of innate immunity.

Molecular mechanisms of NF-κB activation induced by bacterial lipopolysaccharide through Toll-like receptors

Septic shock, caused by exaggerated host responses to various microbial products typified by lipopolysaccharide (LPS), remains the leading cause of death in trauma patients. Gaining insight into the nature of host interactions with LPS will certainly facilitate attempts to develop effective anti-sepsis drugs. Tremendous progress has been made during the past few years in understanding the mechanisms of pathogen-induced host responses. Toll-like receptor (TLR) 4 and 2 have been implicated as major receptors for signaling initiated by LPS and many other microbial products following their binding to CD14. In addition, many signaling intermediates involved in LPS-induced cell activation, particularly activation of the transcription factor NF-kappaB, have been identified and characterized. Further investigations with these molecules will certainly reward us with more effective therapeutic drugs to treat septic shock as well as many other inflammatory and infectious disorders.

Identification and Functional Characterization of Three Chicken Cathelicidins with Potent Antimicrobial Activity

Cathelicidins comprise a family of antimicrobial peptides sharing a highly conserved cathelin domain. Here we report that the entire chicken genome encodes three cathelicidins, namely fowlicidin-1 to -3, which are densely clustered within a 7.5-kb distance at the proximal end of chromosome 2p. Each fowlicidin gene adopts a fourexon, three-intron structure, typical for a mammalian cathelicidin. Phylogenetic analysis revealed that fowlicidins and a group of distantly related mammalian cathelicidins known as neutrophilic granule proteins are likely to originate from a common ancestral gene prior to the separation of birds from mammals, whereas other classic mammalian cathelicidins may have been duplicated from the primordial gene for neutrophilic granule proteins after mammals and birds are diverged. Similar to ovine cathelicidin SMAP-29, putatively mature fowlicidins displayed potent and salt-independent activities against a range of Gram-negative and Gram-positive bacteria, including antibiotic-resistant strains, with minimum inhibitory concentrations in the range of 0.4-2.0 μm for most strains. Fowlicidin-1 and -2 also showed cytotoxicity, with 50% killing of mammalian erythrocytes or epithelial cells in the range of 6-40 μm. In addition, two fowlicidins demonstrated a strong positive cooperativity in binding lipopolysaccharide (LPS), resulting in nearly complete blockage of LPS-mediated proinflammatory gene expression in RAW264.7 cells. Taken together, fowlicidin-1 and -2 are clearly among the most potent cathelicidins that have been reported. Their broad spectrum and salt-insensitive antibacterial activities, coupled with their potent LPS-neutralizing activity, make fowlicidins excellent candidates for novel antimicrobial and anti-sepsis agents.

Structural determinants of host defense peptides for antimicrobial activity and target cell selectivity

Antimicrobial host defense peptides (HDPs) are a critical component of the innate immunity with microbicidal, endotoxin-neutralizing, and immunostimulatory properties. HDPs kill bacteria primarily through non-specific membrane lysis, therefore with a less likelihood of provoking resistance. Extensive structure-activity relationship studies with a number of HDPs have revealed that net charge, amphipathicity, hydrophobicity, and structural propensity are among the most important physicochemical and structural parameters that dictate their ability to interact with and disrupt membranes. A delicate balance among these factors, rather than a mere alteration of a single factor, is critically important for HDPs to ensure the antimicrobial potency and target cell selectivity. With a better understanding of the structural determinants of HDPs for their membrane-lytic activities, it is expected that novel HDP-based antimicrobials with minimum toxicity to eukaryotic cells can be developed for resistant infections, which have become a global public health crisis.

Butyrate Enhances Disease Resistance of Chickens by Inducing Antimicrobial Host Defense Peptide Gene Expression

Host defense peptides (HDPs) constitute a large group of natural broad-spectrum antimicrobials and an important first line of immunity in virtually all forms of life. Specific augmentation of synthesis of endogenous HDPs may represent a promising antibiotic-alternative approach to disease control. In this study, we tested the hypothesis that exogenous administration of butyrate, a major type of short-chain fatty acids derived from bacterial fermentation of undigested dietary fiber, is capable of inducing HDPs and enhancing disease resistance in chickens. We have found that butyrate is a potent inducer of several, but not all, chicken HDPs in HD11 macrophages as well as in primary monocytes, bone marrow cells, and jejuna and cecal explants. In addition, butyrate treatment enhanced the antibacterial activity of chicken monocytes against Salmonella enteritidis, with a minimum impact on inflammatory cytokine production, phagocytosis, and oxidative burst capacities of the cells. Furthermore, feed supplementation with 0.1% butyrate led to a significant increase in HDP gene expression in the intestinal tract of chickens. More importantly, such a feeding strategy resulted in a nearly 10-fold reduction in the bacterial titer in the cecum following experimental infections with S. enteritidis. Collectively, the results indicated that butyrate-induced synthesis of endogenous HDPs is a phylogenetically conserved mechanism of innate host defense shared by mammals and aves, and that dietary supplementation of butyrate has potential for further development as a convenient antibiotic-alternative strategy to enhance host innate immunity and disease resistance.

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.

Structure–activity relationships of fowlicidin‐1, a cathelicidin antimicrobial peptide in chicken

Cationic antimicrobial peptides are naturally occurring antibiotics that are actively being explored as a new class of anti‐infective agents. We recently identified three cathelicidin antimicrobial peptides from chicken, which have potent and broad‐spectrum antibacterial activities in vitro (Xiao Y, Cai Y, Bommineni YR, Fernando SC, Prakash O, Gilliland SE & Zhang G (2006) J Biol Chem281, 2858–2867). Here we report that fowlicidin‐1 mainly adopts an α‐helical conformation with a slight kink induced by glycine close to the center, in addition to a short flexible unstructured region near the N terminus. To gain further insight into the structural requirements for function, a series of truncation and substitution mutants of fowlicidin‐1 were synthesized and tested separately for their antibacterial, cytolytic and lipopolysaccharide (LPS)‐binding activities. The short C‐terminal helical segment after the kink, consisting of a stretch of eight amino acids (residues 16–23), was shown to be critically involved in all three functions, suggesting that this region may be required for the peptide to interact with LPS and lipid membranes and to permeabilize both prokaryotic and eukaryotic cells. We also identified a second segment, comprising three amino acids (residues 5–7) in the N‐terminal flexible region, that participates in LPS binding and cytotoxicity but is less important in bacterial killing. The fowlicidin‐1 analog, with deletion of the second N‐terminal segment (residues 5–7), was found to retain substantial antibacterial potency with a significant reduction in cytotoxicity. Such a peptide analog may have considerable potential for development as an anti‐infective agent.

Fowlicidin‐3 is an α‐helical cationic host defense peptide with potent antibacterial and lipopolysaccharide‐neutralizing activities

Cathelicidins are an important family of cationic host defense peptides in vertebrates with both antimicrobial and immunomodulatory activities. Fowlicidin‐1 and fowlicidin‐2 are two newly identified chicken cathelicidins with potent antibacterial activities. Here we report structural and functional characterization of the putatively mature form of the third chicken cathelicidin, fowlicidin‐3, for exploration of its therapeutic potential. NMR spectroscopy revealed that fowlicidin‐3 comprises 27 amino‐acid residues and adopts a predominantly α‐helical structure extending from residue 9 to 25 with a slight kink induced by a glycine at position 17. It is highly potent against a broad range of Gram‐negative and Gram‐positive bacteria in vitro, including antibiotic‐resistant strains, with minimum inhibitory concentrations in the range 1–2 µm. It kills bacteria quickly, permeabilizing cytoplasmic membranes immediately on coming into contact with them. Unlike many other host defense peptides with antimicrobial activities that are diminished by serum or salt, fowlicidin‐3 retains bacteria‐killing activities in the presence of 50% serum or physiological concentrations of salt. Furthermore, it is capable of suppressing lipopolysaccharide‐induced expression of proinflammatory genes in mouse macrophage RAW264.7 cells, with nearly complete blockage at 10 µm. Fowlicidin‐3 appears to be an excellent candidate for future development as a novel antimicrobial and antisepsis agent, particularly against antibiotic‐resistant pathogens.