Immunol. 3, 569–581 (2003).8. Yednock, T. et al. Nature 356, 63–66 (1992). 9. Engelhardt, B., wolburg-Buchholz, K. & wolburg, H.

Microsc. Res. Tech. 52, 112–129 (2001). 10. Sospedra, M. & Martin, R. Annu. Rev. Immunol. 23,

683–747 (2005).11. Bettelli, E. et al. J. Exp. Med. 200, 79–87 (2004).12. Panitch, H.S., Hirsch, R.L., Haley, A.S. & Johnson, K.P.

Lancet 1, 893–895 (1987).13. willenborg, D.O., Fordham, S., Bernard, C.C., Cowden,

w.B. & Ramshaw, i.A. J. Immunol. 157, 3223–3227 (1996).

14. Gran, B. et al. J. Immunol. 169, 7104–7110 (2002).15. Haak, S. et al. J. Clin. Invest. 119, 61–69 (2009).16. Ke, Y. et al. J. Immunol. 182, 3183–3190 (2009).

nature immunology volume 10 number 5 may 2009 455

Crohn’s disease-associated Nod2 mutants reduce IL10 transcriptionDana J Philpott & Stephen E Girardin

The 3020insC mutation in Nod2 is associated with Crohn’s disease, but how it influences disease pathogenesis is unknown. A new study shows that the 3020insC mutant protein fails to activate a key transcription factor that drives interleukin 10 expression, resulting in reduced production of this anti-inflammatory cytokine.

Dana J. Philpott is in the Department of

Immunology and Stephen e. Girardin is in

the Department of Laboratory Medicine and

Pathobiology at the University of Toronto, Toronto,

Ontario, Canada.

e-mail: dana.philpott@utoronto.ca

Crohn’s disease, a chronic inflammatory disorder that can affect the entire gas-

trointestinal tract1, is generally believed to result from an inappropriate immune reac-tion to the normal commensal flora of the intestine. The most persuasive argument for this conclusion is that in many mouse models of inflammatory bowel disease, regardless of the underlying cause, intestinal inflammation does not develop if the ani-mals are housed under germ-free conditions. Moreover, there is some improvement in patients who take antibiotics or have intes-tinal shunts, which divert intestinal contents away from the inflamed areas. The molecu-lar basis for this overexuberant response to commensal flora is not understood, but the gene encoding nucleotide oligomeriza-tion domain 2 (Nod2) is present within a previously described Crohn’s ‘hot-spot’ on chromosome 16 called IBD1, and three Nod2 coding region polymorphisms are associated with increased susceptibility to disease2,3. In this issue of Nature Immunology, Noguchi et al. find that some of these mutations—specifically, those in the leucine-rich repeat (LRR) domain of Nod2—result in suppres-sion of the production of the immunosup-pressive cytokine interleukin 10 (IL-10).

One of these polymorphisms is a frame-shift mutation resulting from an insertion of a cytosine at nucleotide position 3020 in Nod2 (3020insC). The nucleotide insertion generates a premature stop codon, resulting in a truncated protein lacking part of the last

LRR near the C terminus of the protein. The relative risk for developing Crohn’s disease in individuals bearing a homozygous frame-shift mutation in Nod2 has been calculated to be approximately 30 times that of nor-mal individuals. Moreover, these individuals show a more severe disease phenotype, with increased risk of ileal stenosis and the need for surgical intervention4.

Since the discovery of the link between Nod2 and Crohn’s disease, many studies have focused on trying to understand how mutations in Nod2 influence Crohn’s dis-ease development. Nod2 is an Apaf-like molecule with a distinct domain organi-zation comprising two N-terminal cas-pase-activating and recruitment domains (CARDs), a central nucleotide binding site (NBS) and a C-terminal series of LRRs5. The domain architecture of Nod2 is similar to plant R (for resistance) proteins, which mediate defense against invading patho-gens. Indeed, Nod2, a member of a family of cytosolic innate immune molecules called NBS-LRR receptors (NLRs), is also involved in host defense. Comprising 22 members so far, the NLR family includes proteins that are involved in sensing both microbe- associated and danger-associated molecular patterns within cells and commencing innate immune responses6.

Nod2 responds to a motif called muramyl dipeptide (MDP), which is found in the cell wall of Gram-positive and Gram-negative bacteria7,8 and is a component of peptido-glycan. It is not yet known how Nod2 senses MDP; indeed, no study so far has been able to show whether MDP binds directly to Nod2. On the basis of studies in the Toll-like receptor field and plant R proteins, it has been postulated that the LRR region is likely to represent the site at which ligand sens-

ing occurs, either directly or through a co-receptor. MDP sensing is thought to unfold Nod2, leading to oligomerization through the NBS domain. This process exposes the CARDs, which then provide a platform for the recruitment of Rip2, a CARD-containing signaling kinase that triggers the NF-κB pathway5. Rip2 also activates the p38 and Erk MAP kinases upon MDP stimulation9. These inflammatory pathways lead to the transcrip-tion and expression of several genes, many of which are proinflammatory or provide some defensive function.

In relation to Crohn’s disease, the 3020insC mutation in Nod2 results in a protein prod-uct that, when expressed in vitro, no longer reacts to MDP to drive NF-κB signaling7,8. These findings are supported by studies showing that when cells are isolated from the blood of humans with Crohn’s disease who are homozygous for the 3020insC mutation and treated in vitro with MDP, they do not produce certain cytokines in response to this bacterial product10. A key point, however, is that no study so far has proven that a mutated Nod2 protein is actually present in tissues from subjects with Crohn’s disease. It is pos-sible that this mutation results in an unstable protein product. If this is the case, then the Nod2-deficient mouse is an acceptable model for the disease. However, if the protein is present, it might have ligand-independent functions. Indeed, the paper by Noguchi et al. supports the latter possibility11.

Over the years, much research has gone into trying to unravel the apparent paradox: the 3020insC mutation in Nod2 results in a protein product incapable of responding to a bacterial ligand, yet Crohn’s disease results in overt inflammation that probably is trig-gered by bacterial products. One explana-tion that might help resolve this paradox

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relies on the observation that cells or tissues isolated from Crohn’s disease patients show depressed IL-10 production12. IL-10 is pro-duced by various hematopoietic cells, includ-ing macrophages, dendritic cells and T cells, and acts to contain and suppress inflamma-tory responses and thereby to downmodulate adaptive immune responses and minimize tissue damage in response to microbes. It seems that enhancing IL-10 production, which can occur after treatment with certain probiotic bacteria, helps to calm inflamma-tion in Crohn’s disease12. Moreover, reduced intestinal colonization with an IL-10-promoting member of the commensal flora, Fecalibacterium prausnitzii, is associated with enhanced risk of recurrence of Crohn’s dis-ease after surgical resection13. Noguchi et al. shed light on why IL-10 expression is affected in Crohn’s disease.

Noguchi et al. begin with the observation that bacterially induced IL-10 production is dampened in primary human monocytes from subjects with Crohn’s disease having the 3020insC Nod2 mutation. Although

actual suppression of IL-10 production is not clear from their studies with these cells, synergy between Toll-like receptors (TLRs) and Nod2 is clearly lost. Consistent with this finding, several previous studies have shown a synergistic production of cytokines upon coactivation of TLR2 and Nod2, which is lost in the 3020insC mutants. Transduction of human monocytes with a cDNA encoding the 3020insC mutant Nod2 protein blocks basal transcription and expression of IL-10, but leaves other cytokines, such as IL-1β, unaf-fected. Expression of the 3020insC form of Nod2 blocks IL10 promoter activity as mea-sured from a luciferase reporter. Notably, two other Crohn’s disease–associated Nod2 vari-ants, R702W and G908R, similarly inhibit IL10 promoter activity. The 3020insC Nod2 mutant has no affect on the activity of mouse Il10 reporter constructs and, similarly, the equivalent mutation made in the mouse Nod2 gene, referred to as 2939insC, does not affect human IL10 transcription. Taken together, these findings demonstrate that the human 3020insC mutant of Nod2 acts spe-

cifically to block the human IL10 promoter (Fig. 1). Moreover, it may explain why the 2939insC mouse mutation, when knocked into the mouse Nod2 locus, does not behave like the human 3020insC mutant with regard to proinflammatory cytokine production14.

Noguchi et al. show that heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) binds constitutively to the IL10 promoter. When 3020insC is expressed in cells, the binding of hnRNP A1 to IL10 is reduced. Exogenously expressed hnRNP A1 stimulates IL10 promoter activity, and knocking down hnRNP A1 expression in primary human monocytes reduces lipopolysaccharide- stimulated production of IL-10. Convincingly, through chromatin immunoprecipitation analysis, the authors show reduced binding of hnRNP A1 to the IL10 promoter region in humans with the 3020insC Nod2 genotype, who are highly deficient in IL-10 production.

But how does the presence of the 3020insC mutation in Nod2 result in reduced tran-scription factor binding at the IL10 pro-moter? Noguchi et al. found that wild-type Nod2 forms a trimeric complex in the cyto-plasm with hnRNP A1 as well as active, phosphorylated p38, and it appears that p38 is able to serine-phosphorylate hnRNP A1. This phosphorylation event is required for cleavage of hnRNP A1 into its active form, which can then translocate to the nucleus to act on the IL10 promoter (Fig. 1). However, the 3020insC Nod2 mutant protein interacts with p38 but not with p38hnRNP A1. The lack of this interaction reduces the presence of phosphorylated hnRNP A1 in the nucleus, thereby affecting basal transcription at the IL10 promoter. Interestingly, the mouse 2939insC Nod2 mutant protein resembles wild-type mouse Nod2 in its influence on the interaction between phospho-p38 and hnRNP A1; this observation provides mech-anistic insight into why the mouse mutant does not affect IL10 transcription.

Taken together, the findings of Noguchi et al. stress an important point with regard to the 3020insC mutation in Nod2 associated with Crohn’s disease: this mutation might be a loss of function or gain of function, depend-ing on the response examined. Indeed, it cer-tainly seems that the mutant protein is unable to sense MDP. One might argue that the phe-notype caused by the 3020insC mutation in Nod2 described by Noguchi et al. also repre-sents a loss of function. Indeed, the 3020insC mutant Nod2 protein is unable to form a tri-molecular complex that leads to the activation of hnRNP A1 and thereby affects the ability of hnRNP A1 to bind to the IL10 promoter. However, the authors’ point is well taken that

456 volume 10 number 5 may 2009 nature immunology

MDP stimulated

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Figure 1 Reduced production of iL-10 in cells expressing Crohn’s disease–associated mutations in Nod2. in cells from humans and mice expressing wild-type Nod2, muramyl dipeptide (MDP) triggers nod2 unfolding, allowing its interaction with Rip2 and the subsequent activation of the nF-κB pathway, which drives proinflammatory cytokine gene transcription. noguchi et al. show that wild-type nod2 forms a trimolecular complex with active, phosphorylated p38 and the transcription factor hnRnP A1. This association drives IL10 transcription during the steady-state and after bacterial stimulation (left panel). The Crohn’s disease–associated 3020insC mutant nod2 (right panel) fails to detect MDP and activate the nF-κB pathway, and does not efficiently interact with hnRnP A1; as a result, hnRnP A1 activation and subsequent transcriptional control of the IL10 promoter is impaired. Reduced iL-10 production is therefore a consequence of this mutation and may contribute to the hyperinflammatory response in the intestine that is characteristic of Crohn’s disease. The equivalent mutation translated to mouse nod2 results in a protein that retains its ability to activate Il10 transcription. These findings provide a possible explanation of why cells isolated from a knock-in mouse with this equivalent mutation do not behave like their human counterparts.

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if the mutant protein is indeed expressed, it might have acquired a new, proinflamma-tory “gain of function” that is distinct from the activity of the wild-type version of Nod2. Finally, these studies bring ideas for possible treatments for Crohn’s disease: targeting hnRNP A1 directly to influence its ability to drive IL10 transcription might bypass the block erected by mutant Nod2.

1. Xavier, R.J. & Podolsky, D.K. Nature 448, 427–434 (2007).

2. Hugot, J.P. et al. Nature 411, 599–603 (2001).3. Ogura, Y. et al. Nature 411, 603–606 (2001).4. Seiderer, J. et al. Inflamm. Bowel Dis. 12, 1114–1121

(2006).5. Ogura, Y. et al. J. Biol. Chem. 276, 4812–4818

(2001).6. Franchi, L., warner, n., Viani, K. & nunez, G. Immunol.

Rev. 227, 106–128 (2009).7. Girardin, S.E. et al. J. Biol. Chem. 278, 8869–8872

(2003).

8. inohara, n. et al. J. Biol. Chem. 278, 5509–5512 (2003).

9. Park, J.H. et al. J. Immunol. 178, 2380–2386 (2007).10. netea, M.G. et al. J. Biol. Chem. 280, 35859–35867

(2005).11. noguchi, E. et al. Nat. Immunol. 10, 471–479

(2009).12. Baumgart, D.C. & Sandborn, w.J. Lancet 369, 1641–

1657 (2007).13. Sokol, H. et al. Proc. Natl. Acad. Sci. USA 105, 16731–

16736 (2008).14. Maeda, S. et al. Science 307, 734–738 (2005).

nature immunology volume 10 number 5 may 2009 457

The Foxo and the hound: chasing the in vivo regulation of T cell populations during infectionElia D Tait & Christopher A Hunter

T cell expansion and contraction during the immune response to pathogens are regulated by a wide variety of cell-intrinsic and cell-extrinsic factors. A new study identifies a role for CTLA-4 signaling and activation of the Foxo3 transcription factor in modulating T cell populations.

elia D. Tait and Christopher A. Hunter are in the

Department of Pathobiology, School of veterinary

Medicine, University of Pennsylvania, Philadelphia,

Pennsylvania, USA.

e-mail: chunter@vet.upenn.edu

In this issue of Nature Immunology, Dejean et al. provide new insight into the role of the

forkhead box (Fox) transcription factor Foxo3 in limiting the magnitude of T cell responses1. The Fox family of transcription factors par-ticipate in a multitude of biological processes, including the cell cycle, metabolism and the stress response. The activity of proteins in the Foxo subfamily is predominantly regulated by phosphorylation status and subcellular local-ization, but these transcription factors are also subject to a number of post-translational modi-fications, including acetylation and ubiquitina-tion. Growth factors mobilize numerous kinases, including Akt, that phosphorylate Foxo proteins, leading to their export from the nucleus and inactivation. In contrast, stress stimuli mobilize a different set of kinases, notably Jun N-terminal kinase (Jnk), that promote nuclear import of Foxo and the initiation of Foxo-dependent gene regulation programs2. These transcription factors can modulate gene expression by bind-ing promoters at Foxo-binding sites to recruit transcriptional machinery, by competing for promoter binding sites with other factors, and by dynamically regulating interactions between cofactors and various elements of the transcrip-tional machinery3.

In the past decade, immunologists have come to appreciate the importance of the Fox family in coordinating immune responses, particularly

in the context of Foxp3 expression by T regula-tory (Treg) cells. The Foxo subfamily members (in mammals, Foxo1, Foxo3, Foxo4 and Foxo6) have also been shown to influence T cell func-tion. T cell receptor (TCR) and co-receptor ligation modulate the activity of Foxo proteins that upregulate prosurvival programs in the presence of growth factors and initiate apop-totic pathways in the absence of mitogen or cytokine2. In 2004, Pandiyan et al. demonstrated that signaling through the inhibitory receptor cytotoxic T lymphocyte–associated antigen-4 (CTLA-4) protected T helper type 2 (TH2) cells from apoptosis via Foxo3 inactivation and subsequent upregulation of the survival protein Bcl-2 (ref. 4). Consistent with a proapoptotic, regulatory role for Foxo3, Lin et al. reported that Foxo3a antagonizes signaling by the tran-scription factor NF-κB and that mice carrying a mutated Foxo3a allele underwent spontaneous T cell hyperproliferation and inflammation5. In 2007, it was shown that Foxo3a participates in the maintenance of memory CD4+ T cells by coordinating signals from cytokine receptors and the TCR6. Thus, Foxo proteins appear to have an intrinsic role in T cell homeostasis and in limiting T cell activity.

Dejean et al. examined how Foxo3 influences T cell population expansion and contraction during infection by lymphocytic choriomenin-gitis virus (LCMV). In this model, the absence of Foxo3 led to an exaggerated antigen-specific T cell accumulation, with largely normal con-traction1. Whereas a previous study had sug-gested that Foxo3 was “largely dispensable”5 in modulating T cell apoptosis and was involved exclusively in spontaneous T cell population

expansion, Dejean et al. showed that antigen-specific T cells in Foxo3-deficient mice had enhanced expression of the prosurvival factor Bcl-2 and decreased binding to the apopto-sis marker annexin-5. Notably, the expansion of T cell populations observed in the Foxo3-deficient mice was not T cell intrinsic and was instead dependent on increased production of the cytokine interleukin-6 (IL-6) by Foxo3-deficient dendritic cells (DC)1. IL-6 has long been regarded as proinflammatory, and it also has a strong prosurvival effect on activated T cells7. Blockade of IL-6R α-chain restored the magnitude of the LCMV-driven T cell response in the Foxo3-deficient environment to a level comparable to that observed in wild-type mice. Finally, Dejean et al. showed that CTLA-4 liga-tion induced nuclear localization and activa-tion of Foxo3 and inhibited the production of IL-6 in DCs in vitro1. These findings provide a previously unsuspected mechanism by which CTLA-4–expressing T cells can limit their own survival; by initiating Foxo3 activation and nuclear import in DCs, T cells modulate DC cytokine production and thereby influence their own viability (Fig. 1).

These findings illustrate the importance of the interactions that take place between DCs and T cells in regulating the expansion and contraction of T cell populations during infection. In addition, this work identifies a new role for Foxo3 in modulating this pro-cess. Understanding how T cell responses are regulated and maintained during infection has obvious implications for vaccine develop-ment. To this end, multiple DC–T cell interac-tions that result in the activation of these two

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