Vertebrate innate immunity resembles a mosaic of invertebrate immune responses

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Vertebrate innate immunity resembles a mosaic of invertebrate immune responses Michel Salzet Research on the innate immune response of mammals has revealed similarities with the invertebrate immune system. Thus, insects have developed an acute response resembling that seen in humans, implicating similar effectors, receptors and regulation of gene expression. Mussels have developed intracellular phagocytosis resembling that seen in mammalian neutrophils, using cationic antibacterial peptides in phagolysosomes. Leeches, like amphibians, contain antibacterial peptides and immune stimulators that derive from the processing of neuropeptide precursors. This pattern of similarities suggests that the vertebrate innate immune response resembles a patchwork of those responses seen in several invertebrate models.

Over more than three billion years, bacteria have developed a variety of survival mechanisms that have led to the appearance of antibiotic-resistant bacteria1. This raises the question: how are bacteria kept in check by multicellular organisms? Research on the immune systems of invertebrates, such as insects, has shown that they have developed a battery of natural antibacterial peptides, such as defensin and cecropins2,3, as part of their innate response to bacteria. In addition, antibacterial peptides have been identified in the glandular cells of amphibian skin (e.g. magainins, dermaseptins and ranalexin), humans (e.g. defensins, hadrurin and hepcidin) and plants [e.g. thionins, snaking-1 and antifungal peptides from seeds of Phytolacca americana (PAFP-s)], reflecting the universal nature of such natural defense mechanisms4–9. However, although these antibacterial peptides are present throughout the living kingdom, it is interesting to see how they have evolved. Human antimicrobial responses

In humans, some antimicrobial peptides are produced by the epithelial cells that line the respiratory, gastrointestinal and urogenital tracts and the skin9. Epithelial granulocytes of the small intestine

contribute to the barrier function of the gastric mucosa by the apical release of granules containing a variety of antimicrobial products, including human α-defensin-5 and -6 (Ref. 5). Other similar peptides are found in the glandular secretions that moisten and lubricate such surfaces2. Others, such as human β-defensins, are abundant in certain migratory phagocytic cells that can surround, ingest and kill microbial invaders9. Invertebrate immune responses

Insects, such as Drosophila, respond to septic injuries by rapidly synthesizing antimicrobial peptides (Fig. 1). These peptides are predominantly produced in the fat body; they are then secreted into the hemolymph and participate in a systemic response10. Seven distinct antimicrobial peptides (plus isoforms) have been described for Drosophila. Interestingly, they appear to have distinct target specificities, and induction of the expression of the various peptides depends on the type of infectious agent. Fungal infection, for example, results in a strong induction of the antifungal peptide drosomycin, whereas the antibacterial peptides drosocin and diptericin are only weakly induced11. Conversely, challenge with Gram-negative bacteria strongly induces the antibacterial peptide genes, but has a less marked effect on drosomycin expression11. Drosophila can discriminate between various groups of microorganisms and mount a somewhat adaptive immune response12. The gene Spaetzle codes for a secreted protein of the cysteine-knot family of growth factors, which is activated by proteolytic cleavage12. The processed Spaetzle product is thought to bind to and activate the transmembrane receptor Toll, although direct interaction between the two proteins has not been reported to date. Toll activation is transduced through the adapter molecule Tube and the serine/threonine kinase Pelle, and leads to the phosphorylation and subsequent degradation of the inhibitor Cactus12.

Cactus degradation frees Dif (a member of the Rel family of transcription factors), which translocates to the nucleus, where it is thought to bind to and activate the Drosomycin promoter13. The same genetic analysis revealed that expression of the antibacterial peptides drosocin and diptericin is independent of Toll12. The discovery of the key role played by Toll in the Drosophila host defense led to the description of the first mammalian Toll homolog, now referred to as the Toll-like receptor 4 (TLR4). Two such homologs, TLR2 and TLR4, were shown to play crucial roles in vertebrate innate immunity against bacteria13. TLR2 was shown to play a parallel role in response to peptidoglycan derived from the Gram-positive bacterial cell wall12. Eight additional Toll-related genes (Toll, Toll-3–Toll-8, as well as 18-wheeler), are present in the Drosophila genome. Two of these genes, Toll-3 and Toll-4, are expressed at low levels; by contrast, Toll-6, -7 and -8 are expressed at high levels during embryogenesis and molting, suggesting that Toll and 18w are involved in development13. As well as the Toll pathway, two other pathways more specific for bacterial infection have been demonstrated in Drosophila, namely, imd and 18w (Fig. 1), reflecting the diversity and specificity of responses to pathogens in insects. Epithelial immune responses

Recent studies on antimicrobial peptides from Drosophila have shown that a variety of epithelial tissues in direct contact with the external environment can express the antifungal Drosophila peptide drosomycin, suggesting that a local response to infections is affecting these barrier tissues12. The imd gene in Drosophila plays a crucial role in the activation of this local response to infection. Drosomycin expression is regulated by the Toll pathway during the systemic response, but is regulated by imd in the respiratory tract, thus demonstrating the existence of distinct regulatory mechanisms for local and systemic induction of antimicrobial peptide

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Fig. 1. A simple model of the regulation of Drosophila antimicrobial genes. Seven antimicrobial peptides, as well as their genes, (Drosomycin, Defensin, Metchnikowin, Cecropin, Attacin, Diptericin and Drosocin) have been identified in Drosophila; research on these genes has given rise to the following scheme, which is similar to that found in mammals. Infections by different types of pathogen (e.g. bacteria or fungi) are discriminated by specific receptors and gene activation. The Drosomycin gene is activated by fungi, through Toll receptors; the pathogen-associated molecular patterns (PAMPs) from fungi are recognized by zymogens and interactions between the two stimulate a proteolytic cascade. One of the proteases involved (Spn43Ac) cleaves the cytokine precursor pro-spaetzle to release the active molecule Spaetzle, which then activates the Toll receptor. In this fungal-response pathway, Drosomycin and Defensin genes seem be activated by Dif or Dorsal (dl) homodimers30. By contrast, in response to bacterial infection, Relish (Rel) is probably involved in Drosomycin gene expression. However, both bacteria and fungi activate Attacin and Cecropin genes efficiently through the 18-wheeler (18w) receptor, and the expression of these genes seems to be controlled by a heterodimer of Rel and Dif. Another gene, Metchnikowin, is also activated by bacteria through Toll or imd pathways31,32. These pathways probably employ homodimers of Dif and Rel in binding to κB sites in the gene promoter region. Finally, Diptericin and Drosocin genes are mainly activated by Gram-negative bacteria, through the imd pathway only. Rel is a crucial transcription factor for the Diptericin gene. Modified, with permission, from Ref. 33. Note that some data cannot be explained by this model, and several alternative models have been suggested by other authors. There is no conclusive evidence to indicate whether homo- or heterodimers of Rel proteins are involved in activating these genes.

genes in Drosophila. These results are in line with those found in other invertebrates, such as the mosquito13 and the bloodfeeding fly Stomoxys calcitrans14, and in humans. Thus, the cathelin-class antimicrobial peptides designated LL-37, human β-defensin-1, -2 and -3, are found in skin and the respiratory tract, where they serve to protect surfaces from infection. This might explain why skin and lung infections with Gram-negative bacteria are so rare5. http://immunology.trends.com

Neuropeptide-derived antibacterial peptides

The presence of antibacterial neuropeptide precursors, such as proenkephalin or prodermaseptins, in both invertebrates and vertebrates further supports the hypothesis that these molecules first evolved in ancestral animals8,15–17 (Fig. 2). Indeed, preprodermaseptins form a group of antimicrobial peptide precursors found in the skin of a variety of frog species8.

Precursors of this family have similar N-terminal preprosequences, followed by markedly different C-terminal domains that correspond to mature antimicrobial peptides. This structure is similar to that of cathelicidins found in mammalian neutrophils4. Some of these peptides are 24–34 amino acids long and form stable, amphipathic α-helices; others are disulfide-linked peptides of 20–46 residues; and yet others are highly hydrophobic and the smallest antimicrobial peptides known to date, being only 10–13 residues in length. All of these peptides can kill many bacteria, protozoa, yeasts and fungi by destroying or permeating the microbial membrane. The majority of the peptides act by disintegrating the bacterial membrane or interfering with membrane assembly, with the exception of drosocin, apidaecin and pyrrhocoricin, which appear to deactivate a bacterial protein in a stereospecific manner. In accordance with their biological function, the membraneactive peptides form ordered structures (e.g. α-helices or β-pleated sheets) and often cast permeable ion-pores18. In frogs of the genus Phyllomedusinae, preprodermaseptins are encoded peptides that include dermorphins and deltorphins, which are D-amino-acid-containing heptapeptides. These are potent and specific agonists of the µ- or δ-opioid receptors. In both invertebrates and mammals, the processing of proenkephalin releases the antibacterial peptides, peptide B and enkelytin, associated in turn with opioid peptides that have immune-related activities15–17. In this scenario, the opioid peptides could stimulate immunocyte chemotaxis and phagocytosis, as well as the secretion of immunostimulatory factors. During this process, the simultaneously liberated proenkephalin fragment, having antibacterial activities, could attack bacteria immediately, allowing time for the immune-stimulating capabilities of opioid peptides to manifest themselves. Such innate immune mechanisms seem to be conserved in the course of evolution. In fact, similar results have been found in patients subjected to a coronary bypass, where peptide B and opioids were detected in large amounts just after skin incision15, reflecting their involvement in the initiation of the inflammatory response (Fig. 1). Hemocyte systemic response

In the Limulus model, it has been demonstrated that hemocytes are

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Fig. 2. Antibacterial peptides released from multiple hormone precursors in the innate immune response. Experimentally induced infection, with bacteria or fungi, provokes the release of the neuropeptides dermaseptin and enkephalin through the processing of hormone precursors. (a) and (b) Enkephalins induce the release of signaling molecules (e.g. cytokines) as well as inducing immunocyte chemotaxis themselves, whereas peptide B and enkelytin (released by proenkephalin processing) exert antibacterial actions, in both vertebrates and invertebrates15–17. (b) In vertebrates, the dermaseptin precursor gives rise to antimicrobial substances such as opioid receptor agonists. Enkephalins stimulate the T helper 2 (Th2) lymphocyte responses (through CD3, coupled to intracellular Ca2+ release), which leads to interleukin-4 (IL-4) release and stimulation of IgG release by B cells. Neuropeptides are also responsible for the activation of macrophages and natural killer (NK) cells, resulting in cell killing and inflammation. Thus, in both cases enkephalins act as immune messengers, so-called cytokines. Abbreviation: +, activation. Modified from Ref. 17.

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concentrations in circulating hemocytes are not increased after bacterial challenge25. Within hours following injection, the migration and adherence of hemocytes that contain the antibacterial peptide mytilin was observed around the injection site26. The cells containing mytilin can engulf bacteria, and subsequently release mytilin into phagolysosomes26. By 24–48 hours after bacterial challenge, increased plasma concentrations of mytilin and mussel defensin were detected25–29. This reflects the hemocytic origin of these peptides and favors their participation in a systemic response, as in insects, but at a later stage27,28 (Fig. 3). Thus, the mytilin model of involvement in the anti-infection process exhibits similarities to that described for humans (HNP), in which neutrophil defensins are stored in azurophilic granules that discharge their contents into phagosomes containing microbes, through the process of phagosome–granule fusion29. Conclusions

IgG

extremely sensitive to microbial substances such as lipopolysaccharides and β-glycans. Indeed, upon stimulation, the hemocytes spontaneously degranulate and release into the extracellular fluid a series of substances involved in immune defense19, including several antimicrobial peptides, such as tachyplesins20, big defensin21 and tachycitin22. These results resemble those found in patients with bacterial infections23,24. In these cases,

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increased plasma levels of human neutrophil α-defensin peptides 1–4 (HNP1–4) have been observed, arguing in favor of a systemic antimicrobial response of these peptides during these pathologies23,24. Antimicrobial human neutrophil-like response

In bivalve mollusks (Fig. 3), acquired results demonstrate that antibacterial

Taken together, these data demonstrate that the antimicrobial response towards pathogens has been conserved in the course of evolution. However, antimicrobial responses are not identical among plants, invertebrates and vertebrates4. The convergent point of many studies over the past two decades is that the vertebrate innate response resembles a mosaic of different invertebrate immune mechanisms towards pathogens. Two questions are now pertinent: how are antimicrobial peptides post-translationally activated; and what role does the nervous system play in the production of antimicrobial peptides and their regulation and release? Acknowledgements

I thank P. Bulet (UPR CNRS 9022, Strasbourg, France) and T. Ganz (University of California at Los Angeles School of Medicine, CA, USA) for their critical reading of the manuscript. This review is dedicated to the memory of André Verbert, Director of the UMR CNRS 111, University of Lille, France. This work was supported, in part, by the Centre National de la Recherche Scientifique, the IFREMER, the ANVAR Nord Pas de Calais, and the NIH Fogarty INT 00045.

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Infection site Late phase (24 – 48 hours); degranulation (systemic response)

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Fig. 3. Mytilus innate immune response. Two waves of response are found in the Mytilus defense mechanism. Constitutive expression of antimicrobial genes can be found in circulating hemocytes, with different antimicrobial peptides being stored in different granules of different hemocytes. Two routes of involvement for these peptides have been demonstrated: an immediate intracellular one, and later systemic release into the plasma24–27. Immediately following bacterial infection, hemocytes containing large granules migrate to the infection site where they are implicated in phagocytosis of bacteria. The hemocytic large granules contain antibacterial substances, such as mytilin, which are discharged into the phagosomes upon phagosome–lysosome fusion. Later in infection, other types of hemocyte, containing smaller granules, are implicated in a systemic response. Upon stimulation with microbial substances, such as lipopolysaccharide, these hemocytes spontaneously degranulate, releasing antibacterial peptides (e.g. defensins and mytilins) into the extracellular fluid. Hemocytes containing large granules are also involved in the late phase systemic response, as increased plasma concentrations of mytilin have been detected 24–48 hours after bacterial challenge. It is not known whether hemocytes containing small granules are involved in the early response. This model is similar to that described for human neutrophils (not shown). After phagocytosis of microbes, neutrophil defensins stored in azurophilic granules are discharged into phagosomes containing microbes, through the process of lysosome–phagosome fusion. During this process some of the defensins are released into the extracellular medium and increased plasma concentrations of neutrophil defensins have been observed later in infection.

References 1 Novak, R. et al. (1999) Emergence of vancomycin tolerance in Streptococcus pneumoniae. Nature 399, 590–591 2 Bulet, P. et al. (1999) Antimicrobial peptides in insects; structure and function. Dev. Comp. Immunol. 23, 329–344 3 Steiner, H. et al. (1981) Sequence and specificity of two antibacterial proteins involved in insect immunity. Nature 292, 246–248 4 Lehrer, R.I. and Ganz, T. (1999) Antimicrobial peptides in mammalian and insect host defence. Curr. Opin. Immunol. 11, 23–27 5 Mallow, E.B. et al. (1996) Human enteric defensins. Gene structure and developmental expression. J. Biol. Chem. 271, 4038–4045 6 Ouellette, A.J. et al. (1994). Mouse Paneth cell defensins: primary structures and antibacterial activities of numerous cryptdin isoforms. Infect. Immun. 62, 5040–5047 7 Porter, E.M. et al. (1997) Broad-spectrum antimicrobial activity of human intestinal defensin 5. Infect. Immun. 65, 2396–2401 http://immunology.trends.com

8 Vouille, V. et al. (1997). Structure of genes for dermaseptins B, antimicrobial peptides from frog skin. Exon 1-encoded prepropeptide is conserved in genes for peptides of highly different structures and activities. FEBS Lett. 91, 27–32 9 Huttner, K.M. and Bevins, C.L. (1999) Antimicrobial peptides as mediators of epithelial host defense. Pediatr. Res. 45, 785–794 10 Imler, J.L. and Hoffmann, J.A. (2000) Signaling mechanisms in the antimicrobial host defense of Drosophila. Curr. Opin. Microbiol. 3, 16–22 11 Meng, X. et al. (1999) Toll receptor-mediated Drosophila immune response requires Dif, an NF-kappaB factor. Genes Dev. 13, 792–797 12 Fallon, P.G. et al. (2001) Primitive Toll signalling: bugs, flies, worms and man. Trends Immunol. 22, 63–66 13 Dimopoulos, G. et al. (1997) Molecular immune responses of the mosquito Anopheles gambiae to bacteria and malaria parasites. Proc. Natl. Acad. Sci. U. S. A. 94, 11508–11513 14 Lehane, M.J. et al. (1997) Midgut-specific immune molecules are produced by the blood-sucking insect Stomoxys calcitrans. Proc. Natl. Acad. Sci. U. S. A. 94, 11502–11507

15 Tasiemski, A. et al. (2000) Proenkephalin Aderived peptides in invertebrate innate immune processes. Mol. Brain Res. 76, 237–252 16 Stefano, G.B. et al. (1998) Enkelytin and opioid peptide association in invertebrates and vertebrates: immune activation and pain. Immunol. Today 19, 265–268 17 Salzet, M. et al. (2000) Crosstalk between nervous and immune systems through the animal kingdom: focus on opioids. Trends Neurosci. 23, 550–555 18 Otvos, L. (2000) Antibacterial peptides isolated from insects. J. Pept. Sci. 6, 497–511 19 Muta, T. and Iwanaga, S. (1996) The role of hemolymph coagulation in innate immunity. Curr. Opin. Immunol. 8, 41–47 20 Shigenaga, T. et al. (1990) Antimicrobial tachyplesin peptide precursor. cDNA cloning and cellular localization in the horseshoe crab (Tachypleus tridentatus). J. Biol. Chem. 1265, 21350–21354 21 Saito, T. et al. (1995) A novel type of limulus lectinL6. Purification, primary structure, and antibacterial activity. J. Biochem. (Tokyo) 117, 1131–1137 22 Kawabata, S. et al. (1996) Tachycitin, a small granular component in horseshoe crab hemocytes, is an antimicrobial protein with chitin-binding activity. J. Biochem. (Tokyo) 120, 1253–1260 23 Shiomi, K. et al. (2000) Establishment of radioimmunoassay for human neutrophil peptides and their increases in plasma and neutrophil in infection. Biochem. Biophys. Res. Commun. 195, 1336–1344 24 Panyutich, A.V. et al. (1993) Plasma defensin concentrations are elevated in patients with septicemia or bacterial meningitis. Lab. Clin. Med. 122, 202–207 25 Mitta, G. et al. (2000) Original involvement of antimicrobial peptides in mussel innate immunity. FEBS Lett. 486, 185–190 26 Mitta, G. et al. (2000) Involvement of mytilins in mussel antimicrobial defense. J. Biol. Chem. 275, 12954–12962 27 Mitta, G. et al. (2000) Differential distribution and defence involvement of antimicrobial peptides in mussel. J. Cell Sci. 113, 2759–2769 28 Mitta, G. et al. (1999) Mussel defensins are synthesised and processed in granulocytes then released into the plasma after bacterial challenge. J. Cell Sci. 112, 4233-4242 29 Raj, P.A. et al. (2000) Large-scale synthesis and functional elements for the antimicrobial activity of defensins. Biochem. J. 347, 633-641 30 Manfruelli, P. et al. (1999) A mosaic analysis in Drosophila fat-body cells of the control of antimicrobial peptide genes by the Rel proteins Dorsal and DIF. EMBO J. 18, 3380–3391 31 Tzou, P. et al. (2000) Tissue-specific inducible expression of antimicrobial peptide genes in Drosophila surface epithelium. Immunity 13, 737–748 32 Onfelt Tingvall, T. et al. (2001) The imd gene is required for local Cecropin expression in Drosophila barrier epithelia. EMBO Rep. 2, 239–243 33 Engström, Y. (1999) Induction and regulation of antimicrobial peptides in Drosophila. Dev. Comp. Immunol. 23, 345–358

Michel Salzet Laboratoire d’Endocrinologie et Immunité des Annélides, ESA 8017 CNRS, SN3, Université des Sciences et Technologies de Lille, 59650 Villeneuve d’ascq, France. e-mail: [email protected]