N E W S A N D V I E W S

“... this other Eden, demi-paradise, this fortressbuilt by Nature for herself against infection...”

—William Shakespeare, King Richard II. Act ii. Sc. 1.

Innate immunity is a primary defense againstmicroorganisms, coordinating rapid, tar-

geted responses that eradicate invadingpathogens and neutralize virulence factors. Thefact that naturally occurring microbes threatenboth reproduction and survival leads logicallyto the suggestion that the genes encoding mo-lecules of the innate immune response are subject to considerable directional selectionpressure. Verification of this idea has awaited ademonstration that standing variation in suchgenes has phenotypic consequences for host-pathogen interactions. In a recent article inScience, Clark and colleagues provide just suchevidence, establishing that natural populationsof the fruit fly Drosophila melanogaster showallelic variation that is linked to their ability tofight off bacterial infection1.

Over the last 10 years, D. melanogaster hasemerged as a favorite model organism for thestudy of innate immunity2. Indeed, work inflies has helped catalyze much of the progressof research in vertebrate innate immunity,beginning with the identification of the Tollreceptor and the discovery of its conservation

in humans3. In mammals, Toll-like receptor(TLR) proteins function in both pathogenrecognition and signal transduction4. TLR pro-teins bind specifically to ‘pathogen-encoded’macromolecules such as components of thebacterial cell wall that are not present in eukary-otes but are highly conserved among classes ofmicroorganisms. After interacting with theseforeign molecules, TLR proteins signal via theNF-κB family of transcription factors to triggeran antimicrobial effector response and to

instruct the adaptive immune response viacytokines and costimulatory molecules5. Thereare substantial similarities in the fly innateimmune pathways, although recognition andsignaling are encoded separately (Fig. 1).Dedicated binding proteins interact withmicrobial cell wall components, initiatingextracellular cascades that mainly activate theToll pathway, for infections with fungi orGram-positive bacteria, or a second system, theimmune deficiency (Imd) pathway, when the

474 VOLUME 5 NUMBER 5 MAY 2004 NATURE IMMUNOLOGY

Steven Wasserman is at the Center for Molecular

Genetics, University of California San Diego, La

Jolla, California 92037-0634, USA.

e-mail: stevenw@ucsd.edu

Nature’s fortress against infectionSteven A Wasserman

Correlative analyses of polymorphism and disease resistance in Drosophila melanogaster refine our understanding ofthe forces that maintain variation in innate immunity loci.

Figure 1 The Toll and Imd pathways of D.melanogaster innate immunity. Cell wallcomponents of infecting bacteria bind to andactivate recognition factors in D. melanogasterbelonging to the peptidoglycan recognitionprotein (PGRP) and Gram-negative bindingprotein (GNBP) families. For Gram-positivepathogens (left), the result is proteolyticactivation of spätzle, the ligand for thetransmembrane receptor Toll. Signaling (dottedline) by Toll directs destruction of cactus, an IκB-related inhibitor, and activation of the drosophilaimmunity factor (DIF), a transcription factorclosely related to the mammalian Rel and NF-κBproteins. Gram-negative bacteria (right) engagedistinct factors to activate the Imd protein, acounterpart of the mammalian RIP kinase. Actingthrough a pathway that includes the fly IKKcomplex, Imd effects cleavage and activation ofrelish, the D. melanogaster counterpart of humanp105. The N-terminal, NF-κB-like domain of relish then directs ‘downstream’ gene expression. Underthe control of the Toll and Imd pathways, DIF and relish induce expression of a broad array of innateimmune effectors, including the antimicrobial peptide genes shown here. Genes such as thoseencoding metchnikowin (Mtk), defensin (Def) and attacin A (AttA) respond to both pathways, whereasothers, such as those encoding immune-induced molecule 1 (IM1) and diptericin A (DptA), arepathway specific. Lazzaro et al.1 assayed sequence polymorphisms in and around the genes encodingfactors shown in blue, as well as the genomic regions encoding homologs of factors shown in green.

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mechanism of caspase 1 activation by TLRs is not known, one possible scenario would be the activation of a TLR3–Trif–RIP1–RAIDD–caspase 1 or TLR4–Trif–RIP1–RAIDD–caspase 1 pathway. Several other pro-teins, including TRAF2 and FADD, interactwith RIP1 in the TNF receptor pathway, andtheir possible involvement in Trif-dependentsignaling will need to be tested.

The unexpected involvement of RIP1 inTLR signaling is reminiscent of the imdpathway in drosophila. Imd is a deathdomain adaptor that is most similar to

mammalian RIP1, although it lacks thekinase domain. The imd pathway controlsthe induction of antibacterial responsesthrough the drosophila NF-κB protein rel-ish. In addition, imd mediates caspase-dependent apoptotic signaling in flies. Thecoupling of the inflammatory and apoptoticsignaling pathways is therefore of ancientorigin, and RIP-like proteins seem to be crit-ically involved in their regulation. Definingthese pathways and their importance interms of the immune response to infectionwill be an important challenge.

1. Meylan, E. et al. Nat. Immunol. 5, 503–507 (2004).2. Yamamoto, M. et al. Science 301, 640–643 (2003).3. Oshiumi, H., Matsumoto, M., Funami, K., Akazawa, T.

& Seya, T. Nat. Immunol. 4, 161–167 (2003).4. Yamamoto, M. et al. J. Immunol. 169, 6668–6672

(2002).5. Hoebe, K. et al. Nature 424, 743–748 (2003).6. Fitzgerald, K.A. et al. Nat. Immunol. 4, 491–496

(2003).7. Sato, S. et al. J. Immunol. 171, 4304–4310 (2003).8. McWhirter, S.M. et al. Proc. Natl. Acad. Sci. USA

101, 233–238 (2004).9. Kelliher, M.A. et al. Immunity 8, 297–303 (1998).10. Newton, K., Sun, X. & Dixit, V.M. Mol. Cell. Biol. 24,

1464–1469 (2004).11. Micheau, O. & Tschopp, J. Cell 114, 181–190 (2003).12. Han, K.J. et al. J. Biol. Chem. 279, 15652–15661

(2004).

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NATURE IMMUNOLOGY VOLUME 5 NUMBER 5 MAY 2004 475

infecting agent is a Gram-negative microbe2.Both the Toll and Imd pathways use D.melanogaster NF-κB family members to acti-vate expression of arrays of antimicrobial pep-tides. These peptides are synthesized in adedicated organ, the adult fat body, and arereleased throughout the animal in thehemolymph, the fluid constituent of the openinsect circulatory system.

Loss-of-function mutations in genesencoding either microbial pattern recognitionproteins or signal transducers lead in generalto severely immunocompromised individu-als. Such mutations, although essential to thesuccessful dissection of the mechanismsunderlying innate immunity, are ill suited forinvestigating how the immune responseevolves. For that purpose, researchers haveinstead examined naturally occurring pheno-typic variation at immunity loci within andacross drosophila species. Based on detailedstudies of polymorphism and divergence, ithas been argued that the observed variation ingenes of the innate immune system can onlybe readily explained as the product of naturalselection6. However, it had not been directlydemonstrated that existing polymorphismreflects differences in the ability of individualsto combat infection.

To determine whether detectable polymor-phism is in fact functional, Lazzaro et al. set outto correlate genotypic variation with resistanceto infection. They began by generating a set of107 lines, each homozygous for chromosome 2derived from a distinct isolate collected in thewild. As all other chromosomes were isogenicacross the set, any differences among the linescould be attributed to chromosome 2 (repre-senting approximately 40% of the genome).Flies from each line were stabbed with a cultureof the Gram-negative bacterium Serratiamarcescens. Infected flies were later collectedand the internal pathogen load was assayed byplating of fly homogenates and then countingof colonies. In parallel, DNA was preparedfrom each line and used to derive sequence forthe genomic regions of chromosome 2 thatencode 21 genes of interest. Many of these genesare known to function in innate immunity; theremainder have sequence similarity to knownimmune factors (but no demonstratedimmune function). The extent of polymor-phism at each site was assayed and statisticalmethods were used to calculate the correlationbetween each polymorphic site and the extentof the bacterial load sustained after infection.

The results of the correlation studies werenotable. Isolates of chromosome 2 drawn fromflies in the wild differed in their effect on theability of individuals to resist S. marcescensinfection. At the extremes of the distribution,

bacterial counts were on average an order ofmagnitude higher or lower than the popula-tion mean. Moreover, the determinants of sus-ceptibility to infection were not spreaduniformly among the genomic regions exam-ined. Several polymorphic regions associatedwith the level of resistance to infection werelinked to loci encoding pattern recognition orsignal transduction factors, whereas no suchlinkage was found for the genes encodingantimicrobial peptides.

These studies provide strong support for theidea that standing variation in innate immuneresponse genes can be the object of selectionrelated to disease resistance. Although thisresult seems reasonable in the context of cur-rent knowledge, well considered models pre-dicted very different scenarios. For example, ithas been suggested that one possible conse-quence of an ‘arms race’ between predatorsand prey, or in this case pathogens and hosts,would be that selection would cause resistancealleles to sweep through the population underattack7. If this occurred, the steady-state levelof polymorphism among resistance loci in theperiods between such sweeps would beexpected to be low. The findings of Lazzaro etal. thus establish important constraints tomodel building for evolution of the innateimmune system and provide evidence againstany schemes that cannot accommodate theongoing interaction of selective forces andextant polymorphism.

While notable, these studies leave manyunanswered questions, of which three areconsidered here. First, are the immune lociassociated with variation in resistance in factresponsible for that variation? Experimentsto address this issue might involve measure-ments of polymorphism at other loci distrib-uted across chromosome 2 or, more directly,phenotypic assays of transgenic flies differingonly in the observed alleles of a single locus.It will also be essential to exploit a naturalroute of infection, because wounding of flies,even in sterile conditions, elicits a distinctdefensive response. Second, is there anyimportance in the finding of functional vari-ation among only a subset of the classes ofloci tested? The antibacterial peptide geneswere the sole effector loci examined in thesestudies, a limitation that may have skewedthe outcome for this class. An elegant seriesof transgenic experiments have demon-strated that whole groups of antimicrobialpeptide loci are functionally redundant whenflies are infected with a single bacterialspecies8. Third, what processes maintain thisvariation? More specifically, how much of thevariation results from various forms of posi-tive selection? To answer such a question it

will be important to assay multiple naturaldrosophila pathogens, as particular infec-tious agents evoke both shared and uniquecomponents of the recognition and responsenetwork underlying innate immunity9,10.

Although pathogens are important ininnate immune evolution, other forces arealso at work. In particular, the close interrela-tionship between defensive and developmen-tal pathways imposes considerable bounds onthe nature and extent of variation in immunefactors. For example, one of the immune fac-tors linked to significant phenotypic variationin the paper by Lazzaro et al. is cactus, the flycounterpart to the NF-κB inhibitor IκB. Ascactus is dispensable for the humoral responseto Gram-negative bacteria, this result is at firstunexpected. However, cactus has many devel-opmental functions, including an essentialfunction in larval hematopoiesis11. It may be,therefore, that the significant variation at thislocus modulates the cellular arm of the innateimmune system, that is, its function in the lin-eage of cells responsible for the phagocyticresponse to infection. Similarly, the involve-ment of many innate immune genes inembryonic axis formation and patterning mayact to limit variation at these loci.

Two other areas of ongoing investigation arelikely to have a considerable effect on inquiriesinto evolution of innate immune function. Thefirst is the genomic sequencing of one half-dozen or more drosophila species. The secondis the generation and consolidation of largeamounts of microarray data from organismsinfected with a broad range of pathogens.Together, these two lines of experimentationshould provide a much more detailed pictureof the invertebrate immune system and shouldgreatly facilitate further exploration of themechanism and evolution of host-pathogeninteraction. The understanding growing out ofsuch work is in turn likely to inform attemptsto generate an effective arsenal against theever-changing collection of microbes thatseem at times ‘hell-bent’ on our demise.

1. Lazzaro, B.P., Sceurman, B.K. & Clark, A.G. Science303, 1873–1876 (2004).

2. Brennan, C.A. & Anderson, K.V. Annu. Rev. Immunol.22, 457–483 (2004).

3. Medzhitov, R. & Janeway, C.A., Jr. Curr. Opin.Immunol. 10, 12–15 (1998).

4. Akira, S. Semin. Immunol. 16, 1–2 (2004).5. Beutler, B., Hoebe, K., Du, X. & Ulevitch, R.J.

J. Leukoc. Biol. 74, 479–485 (2003).6. Schlenke, T.A. & Begun, D.J. Genetics 164,

1471–1480 (2003).7. Dawkins, R. & Krebs, J.R. Proc. R. Soc. Lond. B Biol.

Sci. 205, 489–511 (1979).8. Tzou, P., Reichhart, J.M. & Lemaitre, B. Proc. Natl.

Acad. Sci. USA 99, 2152–2157 (2002).9. Lemaitre, B., Reichhart, J.M. & Hoffmann, J.A. Proc.

Natl. Acad. Sci. USA 94, 14614–14619 (1997).10. Hedengren-Olcott, M. et al. J. Biol. Chem. (in the press).11. Qiu, P., Pan, P.C. & Govind, S. Development 125,

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