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The small intestine of a mouse is dark,moist and replete with nutrients—con-

ditions that should make it a paradise forbacteria. Yet very few microbes reside there.What keeps bacteria from populating thesmall intestine? When Eli Metchnikoff con-sidered similar questions in his 1901 mono-graph Immunity in Infective Diseases1, hewrote “The anthrax bacillus, so fatal to miceand guinea pigs, may be swallowed by theseanimals without the slightest danger tothem. To produce generalized anthrax byway of the intestine, it is necessary that theanimals should swallow the spores ofanthrax along with spiny plants, as in theexperiments of Pasteur and his collabora-tors, or along with sand or powdered glass.In these cases, the intestinal lesions served asthe port of entry of the bacillus, the intactmucous membrane preventing their pene-tration. Since diastases (salivary amylase)and the digestive juices are incapable ofaffecting micro-organisms and since certainof the latter perish in the intestines, we mustseek some other cause for their destruction.”In this issue of Nature Immunology, Hornefand colleagues2 describe just such a cause: afamily of potently antimicrobial, smallintestinal peptides called cryptdin-relatedsequence (CRS) peptides.

Hornef et al. identified 17 mRNA speciesencoding 7 different but closely related CRSpeptides in small intestinal tissue fromC57/HeN mice2. Several of these peptideswere purified and were found to be covalentdimers whose CRS components each con-tained 38 amino acids (unless partially

degraded). Overall, 26 residues (68.4%) of theCRS peptides were invariant, including all 9cysteines, 7 prolines and 3 arginines. One cys-teine from each CRS monomer formed anintermolecular disulfide bond with a secondCRS molecule that could be identical to or dif-ferent from its partner. The remaining cys-teines formed disulfide bonds within themonomer. Some dimers showed modestlyenhanced antimicrobial potency relative tothat of the corresponding monomers.

CRS peptides were detected and namedmore than decade ago3,4. Intron analysissuggested that the ancestral cryptdin(intestinal α-defensin) and CRS genesdiverged from a common ancestor long agoand that both genes experienced multiplerounds of reduplication more recently.Although the antimicrobial potential of CRSpeptides was suspected4, the report here byHornef et al.2 is the first to prove it. ‘Prepro’regions of the precursors of CRS and crypt-din peptides show considerable homology,but their mature peptide domains shownone. A similar situation exists in anotherfamily of antimicrobial peptides, the cathe-licidins5. Whereas mature cathelicidin peptides are structurally diverse, theirpropeptides contain a highly conserved, cys-tatin-like domain6 called cathelin, whichmay target the nascent antimicrobial pep-tides and prevent premature proteolysis.

The small intestine produces microbi-cides to prevent bacterial competition fornutrients and to deprive potentialpathogens of an easy portal of entry7.Although ‘Paneth’s cells’ (Fig. 1) weredescribed by Joseph Paneth in 1887 and losttheir apostrophe long ago, our understand-ing of their contribution to small intestinaldefenses is recent8. These cells occupy thelower depths of structures known asLieberkuhn crypts, which are distributed

throughout the small intestine. They con-tain many cytoplasmic granules, and whengastric contents or bacteria enter the smallintestine, Paneth cells release these granulesinto the crypt’s lumen, filling it with anarray of antimicrobial molecules that

Robert I. Lehrer is in the Department of Medicine,

David Geffen School of Medicine at UCLA, Los

Angeles, California 90095, USA.

e-mail: rlehrer@mednet.ucla.edu

Paradise lost and paradigm foundRobert I Lehrer

Antimicrobial peptides are essential effectors of gut immunity. The cryptdin-related sequence peptides represent anewly identified large family of antimicrobial peptides that form dimers to increase diversity.

NATURE IMMUNOLOGY VOLUME 5 NUMBER 8 AUGUST 2004 775

Secretedgranule

"Panethcellins"

CRS peptidesCryptdins

(α-defensins)Secretory PLA2

LysozymeIgA

Storagegranule

Endoplasmicreticulum

Nucleus

Surface

Intestinal lumen

Vill

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.

Figure 1 The small intestine has its ‘ups anddowns’. Nutrients are absorbed by the villi,structures that extend up from the intestinalsurface. Each villus is surrounded by severalcrypts, which descend downward from the surface.Paneth cells, which are found at the bottom of these crypts, contain many large secretorygranules that they discharge into the lumen afterfood or bacteria enter the small intestine. Manycomponents of these granules, including the CRSpeptides described by Hornef et al.2, have potentantimicrobial properties. The complex mixture ofPaneth cell products (‘Panethcellins’) is in pink.‘Panethcellins’ help protect the small intestinefrom microbial infection and colonization.

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Luke A.J. O’Neill is in the Department of

Biochemistry, Trinity College, Dublin, Ireland.

e-mail: laoneill@tcd.ie

How NOD-ing off leads to Crohn diseaseLuke A J O’Neill

Many patients with Crohn disease, an inflammatory bowel disorder, carry mutations in NOD2. The finding thatNOD2 normally dampens Toll-like receptor 2–mediated inflammation may explain this association.

N E W S & V I E W S

include lysozyme, secretory phospholipaseA2, immunoglobulin A and (in mice) up to20 different α-defensins (cryptdins). Nowthat CRS peptides have been shown torapidly kill Gram-positive bacteria (Listeriamonocytogenes and Streptococcus pyogenes)and Gram-negative bacteria (Salmonellatyphimurium, Enterobacter faecalis), theycan be added to the Paneth cell’s ‘antimi-crobial inventory’. If all 7 of the CRS pep-tides described in the report by Hornef et al.were translated, in principle their pairingcould create 28 distinct dimers. PerhapsMetchnikoff anticipated this, for his 1901analysis of small intestinal defenses ended:“The question of the defensive action in thesmall intestine is…far from being settled.The data collected indicate merely that theproblem is a very complex one.”1

The dimeric nature of the CRS peptides isinteresting and unusual, but not unprece-dented. Several other dimeric peptides areknown, including distinctin, from the skinglands of Phyllomedusa distincta (a tree frog);an 11-kilodalton guinea pig peptide; and twopeptides from the hemocytes of a tunicate,Halocynthia aurantium9. In addition, the pro-cessing of θ-defensins (cyclic octadecapep-tides expressed by some nonhuman primates)includes the ‘mix or match’ formation ofdimers10 and forms peptides that vary sub-stantially in antiviral activity11. Nevertheless,CRS peptides provide the first example of afamily that uses dimerization to generate alarge array of antimicrobial molecules.

Why has the murine ‘Department ofDefense’ invested so heavily in developingthese ‘panethcellins’ as ‘weapons of micro-bial destruction’? Some of the threat wasdietary. Although mice sometimes stray

from strict vegetarianism, like all herbivores,they face difficulty in extracting enoughnutrients from a single transit of foodthrough their intestinal tract. Consequently,mice practice ‘hindgut fermentation’ anduse coprophagy to recover the vitamins,amino acids and other nutrients producedby their hindgut flora. However, coprophagyalso presents the murine foregut with theproblem of a large bacterial load. A large anddiverse arsenal of antimicrobial peptidesassists the foregut in handling this microbialchallenge. The natural food of cats (flesh) ismore or less sterile, and the feline smallintestine, like that of other carnivores, lacksPaneth cells. Human diets fall somewherebetween the diets of cats and mice, andhuman Paneth cells express only two α-defensins12,13 and lack CRS peptides.

The report by Hornef et al.2 is comprehen-sive and convincing and uses exemplary tech-niques to identify and characterize the CRSpeptides in the mouse small intestine. Theyused bacteria entrapped in thin agarose gels toidentify and assess the antimicrobial peptides.In vivo, the local control of a bacterial popula-tion requires only that the organisms be killed(or otherwise cleared) more rapidly than theyreplicate. Although conventional broth dilu-tion assays detect molecules that prevent orretard bacterial growth as well as those withbactericidal activity, they have drawbackswhen applied to bactericidal peptides. Forexample, if only one bacterium of the 20,000or so in an assay survives well after exposure tothe peptide, its subsequent overnight growthwill mask the killing of more than 99.9% ofthe organisms in that well. Having the pep-tides diffuse through a gel impregnated withthe test bacteria will reveal any survivors as

microcolonies without hiding the destructionof their microbial ‘siblings’.

The work of Hornef et al.2 also opensmany questions for the future. What is thestructure of these peptides and how do theykill microbes? Do they have antiviral orantiparasitic properties, and how well dothey survive exposure to trypsin and otherdigestive enzymes? It is possible that CRSpeptides may act in synergy with otherantimicrobial components of Paneth cells,such as lysozyme, secretory phopholipase A2and cryptdins. Whether they also exist inwild mice, in other rodents or in other her-bivores remains to be seen. Now thatMetchnikoff ’s questions about the mousesmall intestine have come closer to beinganswered, additional antimicrobial mole-cules should be sought in the human smallintestine. Perhaps human Paneth cells willhave secrets that cannot be divined bystudying the entrails of a mouse.

1. Metchnikoff, E. Immunity in Infective Diseases 423(Cambridge University Press, Cambridge, England,1905).

2. Hornef, M.W. et al. 5, 836–843 (2004).3. Huttner, K.M. & Ouellette, A.J. Genomics 24,

99–109 (1994).4. Ouellette, A.J. & Lualdi, J.C. J. Biol. Chem. 265,

9831–9837 (1990).5. Zanetti, M., Gennaro, R. & Romeo, D. FEBS Lett.

374, 1–5 (1995).6. Yang, Y., Sanchez, J.F., Strub, M.P., Brutscher, B. &

Aumelas, A. Biochemistry 42, 4669–4680 (2003).7. Salzman, N.H., Ghosh, D., Huttner, K.M., Paterson,

Y. & Bevins, C.L. Nature 422, 522–526 (2003).8. Ganz, T. Nat. Immunol. 1, 99–100 (2000).9. Jang, W.S., Kim, K.N., Lee, Y.S., Nam, M.H. & Lee,

I.H. FEBS Lett. 521, 81–86 (2002).10. Leonova, L. et al. J. Leukoc. Biol. 70, 461–464

(2001).11. Yasin, B. et al. J. Virol. 78, 5147–5156 (2004).12. Jones, D.E. & Bevins, C.L. FEBS Lett. 315, 187–192

(1993).13. Jones, D.E. & Bevins, C.L. J. Biol. Chem. 267,

23216–23225 (1992).

776 VOLUME 5 NUMBER 8 AUGUST 2004 NATURE IMMUNOLOGY

What goes wrong in the body when youhave a chronic inflammatory disease?

A root cause for almost all inflammatorydiseases lacking a clear-cut pathogen is still

being sought. In 2001, it was reported that asignificant cohort of individuals with Crohndisease1,2, a debilitating inflammatory con-dition of the gastrointestinal tract, carried amutation in the gene encoding nucleotide-binding oligomerization domain 2 (NOD2).Why this mutation would lead to disease,however, was unclear. Now Watanabe andcolleagues report in this issue of Nature

Immunology3 that one function of NOD2 isto limit the proinflammatory effects medi-ated by Toll-like receptor 2 (TLR2) stimula-tion. The mutant form of NOD2 is unable toinhibit TLR2 signaling, skewing the systemtoward inflammation. The results provide acompelling explanation for why people car-rying the NOD2 mutation might developCrohn disease.

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