Opposing functions of IKK� during acute and chronicintestinal inflammationLars Eckmann*†, Tim Nebelsiek‡, Alexander A. Fingerle‡, Sara M. Dann*, Jorg Mages§, Roland Lang§, Sylvie Robine¶,Martin F. Kagnoff*, Roland M. Schmid‡, Michael Karin†�, Melek C. Arkan‡, and Florian R. Greten†‡

Departments of *Medicine and �Pharmacology, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093; ‡Second Department ofMedicine, Klinikum rechts der Isar, and §Institute of Medical Microbiology, Immunology, and Hygiene, Technical University Munich, 81675 Munich,Germany; and ¶Department of Morphogenesis and Intracellular Signaling, Institut Curie–Centre National de la Recherche Scientifique,75248 Paris Cedex 05, France

Contributed by Michael Karin, August 20, 2008 (sent for review July 25, 2008)

NF-�B is a key transcriptional regulator of inflammatory responses,but also controls expression of prosurvival genes, whose productsprotect tissues from damage and may thus act indirectly in anantiinflammatory fashion. The variable importance of these twodistinct NF-�B-controlled responses impacts the potential utility ofNF-�B inhibition as a treatment strategy for intractable inflamma-tory conditions, such as inflammatory bowel disease. Here, weshow in murine models that inhibition of IKK�-dependent NF-�Bactivation exacerbates acute inflammation, but attenuates chronicinflammatory disease in the intestinal tract. Acute ulcerating in-flammation is aggravated because of diminished NF-�B-mediatedprotection against epithelial cell apoptosis and delayed mucosalregeneration secondary to reduced NF-�B-dependent recruitmentof inflammatory cells that secrete cytoprotective factors. In con-trast, in IL-10-deficient mice, which serve as a model of chronic Tcell-dependent colitis, ablation of IKK� in the intestinal epitheliumhas no impact, yet IKK� deficiency in myeloid cells attenuatesinflammation and prolongs survival. These results highlight thestriking context and tissue dependence of the proinflammatoryand antiapoptotic functions of NF-�B. Our findings caution againstthe therapeutic use of IKK�/NF-�B inhibitors in acute inflammatorysettings dominated by cell loss and ulceration.

NF-�B � colitis � STAT3 � apoptosis � Heat shock protein 70

Inflammation is a fundamental physiological process that protectsthe host against microbial challenges, but can also do great harm

if activated inappropriately or excessively. For example, inflamma-tory bowel disease (IBD) is characterized by persistent inflamma-tion in the colon or small intestine in the apparent absence ofpathogenic microbes. The disease commonly follows a chronicrelapsing course with clinically quiescent periods followed by boutsof severe intestinal inflammation, which are accompanied byabdominal pain, diarrhea, and weight loss. IBD is thought to resultfrom a combination of genetic and environmental factors, but theunderlying causes are probably diverse, depending on specificdisease manifestations and patient subsets (1). In the absence of acomprehensive pathophysiologic understanding of the disease,current therapeutic interventions are largely nonspecific and tar-geted toward attenuating different aspects of the inflammatoryprocess without eliminating its initial triggers.

A key regulator of the inflammatory response is the transcriptionfactor NF-�B, which consists in mammals of homodimers orheterodimers of different NF-�B/Rel proteins (2). NF-�B activatestranscription of numerous target genes, many of which encodecytokines and adhesion molecules that orchestrate the influx andactivation of leukocyte subsets in sites of infection or tissue damage(3). In addition, NF-�B target genes code for proteins that protectcells against apoptosis and necrosis induced by a wide range ofnoxious stimuli (4). In unstimulated cells, NF-�B is retained in thecytoplasm by specific inhibitors, the I�B proteins. Stimulation withmicrobial ligands of Toll-like receptors (TLRs) or the prototypicproinflammatory cytokines, TNF-� or IL-1, induces I�B phosphor-

ylation and ubiquitin-dependent proteasomal degradation resultingin nuclear entry of NF-�B dimers to initiate target gene transcrip-tion. I�B phosphorylation is catalyzed by the I�B kinase (IKK)complex composed of two catalytic subunits, IKK� and IKK�, inconjunction with a scaffolding and regulatory protein, IKK�/NEMO (5). Whereas IKK� is activated by an only limited set ofstimuli, IKK� activation occurs upon receptor-mediated stimula-tion by a broad set of microbial or host-derived ligands (2).Inhibition of IKK� suppresses the production and secretion of theprototypic proinflammatory cytokine TNF-� and attenuates dis-ease in animal models of rheumatoid arthritis, inflammation-induced bone loss, and allergen-induced airway disease (6–9).Furthermore, inhibition of NF-�B by either antisense oligonucle-otides directed against RelA/p65 or a small-molecule inhibitor ofIKK� appeared to ameliorate disease in mouse models of intestinalinflammation (10–12), suggesting that NF-�B-directed therapycould be a valuable novel strategy in IBD therapy. In sharp contrast,however, conditional ablation of IKK� in intestinal epithelial cells(IECs) caused increased inflammation in an acute, chemicallyinduced colitis model (13) and loss of IKK� in IECs causedspontaneous colitis (14). Moreover, recent findings demonstratingan important antiinflammatory function for NF-�B based onsuppression of IL-1� processing and secretion (15) raise concernsabout the therapeutic potential of IKK� inhibitors (16). We un-dertook the present study to resolve these discrepancies and de-termine the role of canonical, IKK�-dependent NF-�B activationin different cell types during acute ulcerating and chronic colitisdriven by immune dysregulation rather than epithelial injury.

ResultsIKK� Is Necessary for Early Healing After Acute Colitis. Loss of IKK�in epithelial cells renders the cells vulnerable to apoptosis afteracute exposure to various stress insults (13, 17, 18). Accordingly, weobserved a protective effect of IEC-IKK� in acute colitis caused bysuppression of epithelial cell death early in disease induction andhence maintenance of epithelial integrity and barrier function (13).Cell culture studies have shown that NF-�B can also controlepithelial cell migration (19), a process that contributes to epitheliallayer healing after acute injury. To determine whether epithelialIKK� has a role in the early healing phase after acute colitis, weadministered dextran sulfate sodium (DSS) in the drinking water tomice lacking IKK� in IECs (Ikk�DIEC mice) and littermate controlsfor 5 days followed by 16 days of regular drinking water (Fig. 1A).Histological analysis carried out 21 days after initiation of DSS

Author contributions: L.E. and F.R.G. designed research; T.N., A.A.F., S.M.D., and M.C.A.performed research; S.R., M.F.K., and R.M.S. contributed new reagents/analytic tools; L.E.,J.M., R.L., M.C.A., and F.R.G. analyzed data; and L.E., M.K., M.C.A., and F.R.G. wrote thepaper.

The authors declare no conflict of interest.

†To whom correspondence may be addressed. E-mail: leckmann@ucsd.edu, mkarin@ucsd.edu, or florian.greten@lrz.tum.de.

© 2008 by The National Academy of Sciences of the USA

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exposure, corresponding to the late healing phase in this colitismodel, demonstrated that IEC-specific deletion of IKK� wasassociated with more severe mucosal inflammation and greaterareas of ulceration in comparison with littermate controls (Fig. 1B–E).

To exclude the possibility that the observed late healing pheno-type might have been caused by increased early damage induced byincreased apoptosis in IKK�-deficient enterocytes (13), we treated

WT mice after completion of DSS administration with a highlyspecific IKK� inhibitor, ML120B (20), or vehicle for 5 days (Fig.1F). The inhibitor was highly effective in blocking NF-�B activationin the inflamed colon (Fig. 1G). Ten days after the start of DSSapplication, corresponding to the beginning of the healing phase,mice were examined histologically. Pharmacological IKK� inhibi-tion, like enterocyte-specific genetic ablation, induced greater areasof ulceration and more severe mucosal inflammation comparedwith vehicle-treated controls (Fig. 1 H–K). These results stronglysupport the notion that IKK� has additional functions in IECsbeyond protection against apoptosis and further demonstrate thatIKK� exerts an overall protective function in acute colitis indepen-dently of the initial damage.

Pharmacological IKK� Inhibition Suppresses Epithelial Expression ofNF-�B and STAT3 Target Genes. To unravel the mechanisms respon-sible for the epithelial healing defect in ML120B-treated animals,we isolated enterocytes and performed a microarray analysis byusing Affymetrix MOE430A 2.0 GeneChips (containing �22,000probe sets that represent �14,000 annotated genes). Mice wereanalyzed on day 7, at which time tissue damage and inflammationis most severe and the process of epithelial healing commences. Wefocused on decreased gene expression that correlated with IKK�inhibition to identify critical protective genes. Expression of �100genes was �2.5-fold decreased in IECs of ML120B-treated micecompared with vehicle-treated controls. More than 30% of theidentified genes represented known NF-�B target genes, includingCxcl1, Cxcl2, Cxcl5, Cxcl9, Cxcl10, Cxcl20, Ccl5, Ccl8, Icam1, andPla2g2a (21, 22). Down-regulation of several of these genes byML120B after DSS treatment was confirmed by real-time PCRanalysis (Fig. 2A). However, the products of these NF-�B targetgenes have proinflammatory functions, so their down-regulationdid not explain the increase in inflammation after ML120Btreatment.

Surprisingly, the microarray analysis revealed a second group ofdown-regulated genes, including Hspa1a, Tgtp, Ifitm1, Igtp, Ifi47,Iigp, H2-Aa, H2-Ab1, and Socs3, whose expression is known to becontrolled primarily by STAT and IRF transcription factors. Of

Fig. 1. IKK� inhibition delays healing in acute ulcerating colitis. (A) Sche-matic overview of the DSS-induced colitis model. (B–E) Histological score (B),size of ulcerations (C), and representative H&E-stained sections (D and E) ofIkk�F/ F and Ikk�DIEC colons during the late healing phase of acute colitis, 21days after the beginning of DSS administration. (F) Schematic overview ofML120B treatment in DSS colitis. (G) EMSA shows inhibition of total NF-�Bbinding activity in whole colonic extracts of WT mice treated with ML120B (80mg/kg, twice a day) for 5 days compared with vehicle-treated controls afterthey had received DSS for 5 days. (H–K) Histological score (H), size of ulcer-ations (I), and representative H&E-stained sections (J and K) of the colon of WTmice left untreated or treated with ML120B for 5 days after the end of DSSadministration. P values were determined by Student’s t test and were con-sidered significant when �0.05. (Magnifications: D, E, J, and K, 20�.)

Fig. 2. IKK� inhibition suppresses NF-�B and STAT3 target gene expression inenterocytes during initiation of healing of acute ulcerating colitis. (A) Expressionof NF-�B and STAT3 target genes. Relative mRNA levels were determined byreal-timePCR in isolated IECs frommice thathadreceivedDSSfor5daysandwereeither left untreated or treated with ML120B for 2 days. Data are mean � SE. *,P � 0.05 by t test. (B) HSP70 expression and activation of STAT3 were determinedby immunoblot analysis in IECs of mice treated as in A. (C) ML120B has no effecton phosphorylation of STAT3 in colon cancer cells treated with IL-6 for 30 min.

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particular interest in this group was Hspa1a, which encodes induc-ible heat shock protein (HSP) 70 whose epithelial expressionprovides protective functions during DSS-induced colitis (23).Immunoblot analysis confirmed nearly complete absence of HSP70expression in enterocytes from ML120B-treated mice after DSSadministration (Fig. 2B). Loss of HSP70 expression was paralleledby decreased activation of epithelial STAT3 (Fig. 2B), a keyregulator of Hspa1a transcription (24). No differences were ob-served in STAT1 phosphorylation (data not shown). The attenu-ation of STAT3 activation was not related to any direct effects ofML120B on epithelial STAT3 signaling (Fig. 2C), consistent with aprevious report that ML120B is a highly specific IKK� inhibitor(20). These results suggest that inhibition of NF-�B indirectlyattenuates epithelial STAT3 signaling, resulting in loss of cytopro-tective HSP70 in IECs.

Inhibition of IKK� Attenuates Inflammatory Cell Recruitment andExpression of Cytoprotective IL-11 and IL-22. To test the hypothesisthat IKK� inhibition reduces mucosal production of STAT3 acti-vators in DSS-induced colitis, we isolated RNA from whole colonicmucosa on day 7 after initiation of DSS administration andexamined expression of known STAT3 activators, including type Iand II IFNs, as well as IL-6 and IL-10 cytokine family members. Noexpression differences were detected in mRNAs coding for IL-6,IL-10, IFN-�, IFN-�, or IFN-� in ML120B-treated and controlmice (Fig. 3A and data not shown). However, ML120B inhibited thecolitis-associated increase in IL-11 and IL-22 mRNAs in wholecolonic mucosa (Fig. 3A). To examine whether IL-11 and IL-22 aredirectly regulated by NF-�B, we examined mRNA expression incontrol and IKK�-deleted bone marrow-derived macrophages (15)before and after LPS stimulation. LPS induced expression of bothIL-11 and IL-22 mRNAs in control macrophages. Ablation of IKK�

did not attenuate this induction but rather enhanced it (Fig. 3B),indicating that NF-�B is not required for induction of IL-11 andIL-22 expression. The expression of two known NF-�B targets, IL-6and IL-10, was inhibited, as expected, in IKK�-deficient macro-phages (Fig. 3B). These data suggest that ML120B did not suppressmucosal IL-11 and IL-22 mRNAs by direct inhibition of their geneexpression.

As an alternative, we considered whether ML120B inhibited therecruitment of cells that produce these cytokines into the mucosa.We therefore assayed the expression of various markers for my-eloid, natural killer (NK), and T cells, including CD11c, F4/80, Gr-1,CD49b, CD4, CD8, and FoxP3. Interestingly, ML120B led to amarked decrease in expression of F4/80 and CD4 after DSSadministration (Fig. 3C), indicating a decrease in macrophages andCD4� T cells in the lamina propria of inhibitor-treated mice. On theother hand, expression of the markers for NK cells, CD8� T cells,and regulatory T cells were not affected, and the neutrophil-specificmarker Gr-1 was more highly expressed in ML120B-treated ani-mals (Fig. 3C). The latter indicates increased neutrophil numbersin the colon after ML120B treatment, which is consistent withreports of blood neutrophilia caused by IKK� inhibition (15, 20).These results, together with the cytokine analyses (Fig. 3 A and B),strongly argue that the ML120B-induced inhibition of IL-11 andIL-22 expression in the mucosa was not due to suppression of thesecytokines in macrophages, but rather related to the failure to recruitthese cells into the mucosa. Our data suggest a circular paracrinemodel in which IKK� inhibition decreases epithelial chemokineinduction in colitis, thereby interfering with recruitment of cells thatexpress the key protective cytokines IL-11 and IL-22 (see Fig. 6).These cytokines are in turn required for activation of epithelialSTAT3 and induction of normal levels of cytoprotective HSP70 inthe epithelium.

Fig. 3. IKK� inhibition prevents IL-11 and IL-22 ex-pression in colonic mucosa and recruitment of criticalinflammatory cells. (A) Relative mRNA levels were de-termined by real-time PCR in whole colonic mucosa ofmice that received DSS for 5 days and were either leftuntreated or treated with ML120B for 2 days. (B) Rel-ative mRNA levels in bone marrow-derived macro-phages of Ikk�F/ F and Ikk�D mice (15) before and afterstimulation with LPS (100 ng/ml) for 4 h. (C) RelativemRNA levels of cell type-specific markers were deter-mined by real-time PCR in whole colonic mucosa ofmice that had received DSS for 5 days and were eitherleft untreated or treated with ML120B for 2 days. Dataare mean � SE. *, P � 0.05 by t test.

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Delayed Loss of Epithelial IKK� Has No Impact on Late Healing AfterAcute Colitis. To further confirm that the initial recruitment ofinflammatory cells is responsible, at least in part, for initiation of thehealing process after acute colitis, we took advantage of a model ofinducible IKK� deletion in the intestinal epithelium. Mice carryinga tamoxifen-inducible Cre transgene under control of the epithe-lium-specific Villin promoter (25) were crossed with floxed Ikk�mice (26). Oral administration of tamoxifen for 5 days causedeffective deletion of IKK� in the small intestinal and colonicepithelium, but not in lamina propria cells or in any of the otherorgans tested (Fig. 4A).

Having established that IKK� can be effectively deleted in atemporally controlled fashion, we induced acute colitis in IKK�-proficient mice by DSS administration for 6 days, followed by 4 dayson regular water (corresponding to the period of maximal inflam-mation, recruitment of immune cells, and the beginning of woundhealing), after which we started tamoxifen treatment for 5 days toinduce epithelial IKK� deletion (Fig. 4B). In contrast to mice withconstitutive IKK� deletion (Fig. 1 B–E), the mice did not exhibitany difference in regard to mucosal inflammation or epithelialhealing 21 days after the start of DSS administration (Fig. 4 C andD). These results suggest that once the critical inflammatory cellsare recruited into the mucosa, they can produce cytoprotectivecytokines, such as IL-11 and IL-22, independent of epithelial IKK�and NF-�B. The data further indicate that epithelial IKK� has two

important physiologic functions in acute colitis, cell-autonomousearly protection against apoptotic cell death (13) and delayedepithelial protection and healing via recruitment of inflammatorycells that release cytoprotective factors.

Loss of IKK� in Macrophages/Neutrophils but Not Epithelial CellsAttenuates Chronic T Cell-Driven Colitis. Although our studies re-vealed an important protective function for IKK� in acute colitis,the observations remained apparently inconsistent with prior stud-ies on the inflammation-promoting function of RelA/p65 (11). Akey difference between our and prior studies was the duration of thecolitis (acute vs. chronic) and the underlying pathogenic mecha-nisms, particularly the involvement of T cells. To bridge the gapbetween the models, we used an injury-independent model of Tcell-driven chronic colitis associated with IL-10 deficiency. IL-10-deficient mice develop spontaneous mucosal inflammation andIEC hyperplasia in the colon over a period of months (27).Inflammation was associated with increased NF-�B activity in theentire colon and isolated IECs (Fig. 5 A and B), suggesting thatcanonical NF-�B activation may also play a role in controlling colitisin this model. To examine this point, we crossed Il10�/� mice withIkk�DIEC mice to yield mice double-deficient for IL-10 and IEC-IKK� (Il10�/�/Ikk�DIEC mice). As controls we used littermates thatlacked IL-10 and had floxed Ikk� but no Cre transgene (Il10�/�/Ikk�F/F mice). Both groups were examined for the occurrence andseverity of spontaneous colitis. Loss of epithelial IKK� had nosignificant impact on the incidence of spontaneous colitis (Fig. 5C)or the severity of mucosal inflammation once animals had devel-oped significant disease (Fig. 5 D–G). Moreover, the time intervalduring which animals lost 20% of their prior maximal body weight,a clinical measure of the acuteness of disease, was the same in allmice independent of their genotype (Fig. 5 H and I). Thus, despitemarked activation of NF-�B in the epithelium in this model ofchronic colitis, its function in IECs was dispensable under theseconditions. By contrast, crossing of Il10�/� mice with Ikk�Dmye micethat lack IKK� selectively in macrophages and neutrophils (13)resulted in significant attenuation of spontaneous colitis (Fig. 5J).

To determine whether key inflammatory factors known to beimportant for pathogenesis in this chronic colitis model are affectedby deletion of IKK� in myeloid cells, we isolated bone marrow-derived macrophages from Il10�/� mice. Macrophages were stim-ulated with LPS in the presence or absence of ML120B, and mRNAlevels for several cytokines and other inflammatory mediators weredetermined. ML120B blocked induction of TNF-�, IL-1�, IL-6,ICAM-1, IL-12p40, and IL-23p19 mRNAs after LPS stimulation(Fig. 5K). Collectively, our data show that, in contrast to acuteinjury-related colitis, the dominant activity of IKK� in chroniccolitis is proinflammatory and mediated by myeloid cells, whereasits epithelial functions have no impact on the course or severity ofinflammation.

DiscussionThis study demonstrates that NF-�B has overall protective func-tions in acute, ulcerating colitis, but promotes inflammation inchronic colitis that is not associated with rapid and extensivemucosal injury. These results help to reconcile apparently discrep-ant information from prior reports on the role of NF-�B incontrolling intestinal inflammation (11, 12). Thus, mice selectivelydeficient for IKK� in IECs exhibit more severe acute colitis (13) andepithelial loss of IKK� caused spontaneous colitis (14), supportingthe notion that IEC IKK� and NF-�B have an important protectiverole that prevents injury-induced mucosal inflammation. Our datademonstrate that the protective functions of IKK� and NF-�Bdominate their overall activity in the intestinal mucosa under acuteinflammatory conditions. Consistent with this interpretation, treat-ment with LPS, which strongly activates NF-�B in epithelial andother mucosal cells, attenuates acute chemical colitis and irradia-tion-induced damage (17, (28). Protection by NF-�B is in part

Fig. 4. Deletion of IKK� after initial recruitment of inflammatory cells has noeffect on healing of acute ulcerating colitis. (A) Selective ablation of IKK� inIECs throughout the entire intestine (duodenum, jejunum, ileum, colon) ofvillin-Cre-ERT2/Ikk�F/ F mice after induction of Cre-recombinase activity bytamoxifen (TAM). (B) Schematic overview of DSS and tamoxifen application.(C and D) Histological score (C) and size (D) of ulcerations 21 days after thebeginning of DSS administration in villin-Cre-ERT2/Ikk�F/ F mice, which hadreceived TAM 5 days after the end of DSS administration.

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mediated by its direct effects on epithelial cell survival (13). Ourresults further suggest that NF-�B also protects against damage ina paracrine manner by up-regulating the expression of chemokinesthat recruit myeloid and T cells (Fig. 6). These cells releasecytoprotective factors such as IL-11 and IL-22, whose protectivecapacity against acute colitis has been demonstrated by directadministration or local gene delivery in WT animals (28, 29).Myeloid cells, in particular, can contribute to protection againstacute colitis, because their depletion exacerbated acute DSS-induced colitis (30). One possible mechanism by which NF-�B-dependent myeloid cell recruitment can mediate epithelial protec-tion and healing is through production of IL-11 and IL-22 and otherfactors that induce epithelial expression of HSP70, which has strongantiapoptotic functions in IECs (31, 32). Indeed transgenic expres-sion of HSP70 in IECs ameliorated acute DSS-induced colitis (23).

Hspa1a, which encodes HSP70, is a STAT3 target gene and does notcontain NF-�B binding sites in its promoter region and is not knownto be induced by IKK activators. However, IL-11 and IL-22 arepotent activators of STAT3 and HSP70 expression, suggesting thatNF-�B can mediate mucosal protection indirectly by recruitingmyeloid and T cells, which produce these cytoprotective cytokines(Fig. 6).

In contrast to acute ulcerating colitis, epithelial IKK� plays noapparent role in chronic colitis driven by immune dysregulation. Inthe IL-10-deficient model of chronic colitis, disease development iscaused by an imbalance of inflammation-causing effector T cellsand inflammation-suppressing regulatory T cells, with antigens ofthe normal microbiota as the driving force (27). Epithelial involve-ment is secondary to the influx of inflammatory cells and ischaracterized mostly by hyperproliferation rather than frank ulcer-ation (33). Epithelial erosions are not a central feature of thepathogenesis and, if at all, are superficial and occur very late in thedisease process. Under these conditions, IEC-NF-�B was activatedbut had no tangible impact on disease progression, possibly becauseof the reduced pathogenetic importance of protection against celldeath. Alternatively, prolonged exposure of epithelial cells toinflammatory mediators may render their protection independentof IKK�/NF-�B, perhaps because other protective pathways areoveractivated. In contrast, IKK� in macrophages and neutrophilspromoted inflammation under the same circumstances, indicatingthat myeloid NF-�B has a key proinflammatory function in thismodel of chronic colitis. These data are consistent with, and canexplain, the overall antiinflammatory effects achieved by adminis-tration of antisense oligonucleotides against NF-�B/RelA in adifferent model of T cell-driven chronic colitis (11).

NF-�B controls the expression of numerous genes encodingchemokines and proinflammatory cytokines and several antiapo-ptotic proteins (34). Our current and prior studies provide strongevidence that the relative physiologic importance of these twomajor groups of target genes, within the same tissue and disease

Fig. 5. Ablation of myeloid but not epithelial IKK�

ameliorates colitis in Il10�/� mice. (A) Increased NF-�Bbinding activity in IECs and lamina propria cells (LP) inIl10�/� mice with clinical signs of disease determined byEMSA. Immunoblot analysis (WB) of �-actin was per-formed to control for using equal amounts of proteins.(B) NF-�B composition determined by a supershift assayin IECs and LP from mice with colitis. (C) Disease-freesurvival of Ikk�F/ F and Ikk�DIEC mice on an IL-10-deficientbackground was followed for 30 weeks after birth. (Dand E) Representative H&E-stained sections of Ikk�F/ F/Il10�/� and Ikk�DIEC/Il10�/� double-mutant mice. (Magni-fication: 20�.) (F and G) Analysis of crypt depths (F) andnumbers of infiltrating cells (G) at the end of the disease-free survival period in Ikk�F/ F/Il10�/� and Ikk�DIEC/Il10�/�

double-mutant mice. (H and I) Time span between age atmaximal body weight and the end of the disease-freesurvival period (H) and degree of weight loss during thelast 2 weeks before the end of disease-free survival (I) ofIkk�F/ F/Il10�/� and Ikk�DIEC/Il10�/� double-mutant mice.(J) Disease-free survival in Ikk�Dmye mice and Ikk�F/ F micelacking IL-10 was followed for 20 weeks. Note that ani-mals in C and J were kept in different animal facilities,which presumably accounts for the differences in dis-ease-free intervals. (K) Real-time PCR analysis of the in-dicated mRNAs in IL-10-deficient bone marrow-derivedmacrophages treated with LPS (100 ng/ml) for the indi-cated times in the presence or absence of ML120B (30�M). Data are mean � SE. *, P � 0.05 by t test.

Fig. 6. Proposed model of IKK� actions in acute ulcerating colitis. DuringDSS-induced colitis, IKK� and NF-�B are activated in IECs, leading to increasedtranscription and secretion of chemotactic cytokines for myeloid and T cells.Recruitment of these cells is important for IKK�-independent mucosal expressionof cytokines, including IL-11 and IL-22, that are cytoprotective for epithelial cellsthrough activation of STAT3 and downstream target genes such as HSP70.

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process, varies depending on the specific cell types involved and theparticular etiologic and pathogenetic mechanisms. Thus, NF-�B-dependent cell survival and chemokine expression is dominant inepithelial cells under acute injury conditions, whereas proinflam-matory functions of NF-�B are decisive in chronic situations ofimmune dysregulation. It should be noted that under most patho-physiologic conditions, including those in IBD patients, these setsof functions are likely to coexist and may even compete with eachother. Therefore, the overall effects of IKK�/NF-�B inhibition indifferent organ systems and disease processes are not easily pre-dictable under all circumstances, but must be carefully establishedfor successful therapeutic applications.

Our results have implications for the potential therapeutic use ofIKK� inhibitors and add to recently raised concerns about pro-longed inhibition of this pathway (15). Although pharmacologicalinhibition of IKK� had been suggested to improve acute colitis (12),our results do not support this notion. One possible explanation forthese differences is the selectivity of the particular inhibitors used,and the possibility that other inflammatory pathways might beaffected nonspecifically. The inhibitor used in our studies, ML120B,displays very high specificity in vivo and its overall effects closelymimic the genetic deletion of IKK� in enterocytes during DSS-induced colitis as well as those observed after IKK� ablation inmyeloid cells in models of sepsis and endotoxic shock (15). Basedon our genetic and pharmacological results, inhibition of IKK� andNF-�B is likely to exacerbate tissue damage during the acute phaseof intestinal inflammation dominated by apoptotic loss of epithe-lium and subsequent ulceration, and would therefore appear to becontraindicated. In contrast, inhibitors of IKK� and NF-�B may bebeneficial in the chronic phase of intestinal inflammation, when therisk of epithelial cell apoptosis and epithelial ulcerations is reducedor entirely absent, especially if such inhibitors are targeted tomyeloid cells.

Materials and MethodsMice. To generate Ikk�DIEC mice, Ikk�F/F mice (26) were crossed to Villin-Cre mice(35) and kept on a C57BL/6;129 background. Ikk�Dmye mice have been described

(13). For enterocyte-specific and temporal ablation of Ikk�, villin-Cre-ERT2 mice(25) were crossed to Ikk�F/F. Deletion was induced by five daily oral administra-tions of 1 mg of tamoxifen in an ethanol/sunflower oil mixture. Il10�/� mice on aC57BL/6 background were obtained from The Jackson Laboratory and inter-crossed with Ikk�DIEC and Ikk�Dmye mice. Occurrence of significant spontaneousdisease, and thereby the end of disease-free survival, in IL-10-deficient mice wasdefined as �20% loss of body weight relative to maximal prior weight. Diseasedmice were euthanized and examined histologically. All animal procedures werereviewed and approved by the Regierung von Oberbayern and the University ofCalifornia at San Diego Institutional Animal Care and Use Committee.

Colitis Induction and Analysis. To induce acute colitis, mice were given 3% DSS(MP Biomedicals) in their drinking water for 5–6 days, followed by regulardrinking water. ML120B, kindly provided by Millenium Pharmaceuticals, wasgiven by oral gavage twice daily in methylcellulose at a concentration of 80mg/kg. Mice were euthanized on the indicated days, and the colon was removed,fixed in paraformaldehyde, and embedded in paraffin. Severity of colitis wasassessed histologically as described (13, 36).

Protein and RNA Analysis. Isolation and culture of bone marrow-derived mac-rophages, RNA extraction, cDNA synthesis, and real-time PCR were performed asdescribed (15). Primer sequences are available on request. Gene expression pro-filing of isolated enterocytes was performed with Affymetrix MOE430A 2.0GeneChipsas reported(37). Isolationof IECs, immunoblots,andEMSAshavebeendescribed (13). The following antibodies were used: anti-IKK� (Imgenex), anti-IKK� (UBI), anti-RelA/p65, anti-p50, anti-c-Rel (Santa Cruz), and anti-�-actin(Sigma).

ACKNOWLEDGMENTS. We thank Birgit Wittig, Kristin Retzlaff, and Lucia Hallfor excellent technical assistance and expert help with the animal studies. Thiswork was supported by National Institutes of Health Grants DK70867, DK35108,and RR17030, the University of California at San Diego Digestive Diseases Re-search Development Center (Grant DK80506), and a Jeannik M. Littlefield–American Association for Cancer Research grant (to M.K.). Further support wasprovided by Wilhelm Sander-Stiftung Grant 2005.146.1 (to M.C.A.) and theDeutsche Forschungsgemeinschaft (Emmy-Noether-Program Grant Gr1916/2–2,SFB 456), Deutsche Krebshilfe Grant 106772, and Fritz-Thyssen-Stiftung Grant10.05.2.168 (to F.R.G.). M.K. is an American Cancer Society Research Professor.

1. Sartor RB (2006) Mechanisms of disease: Pathogenesis of Crohn’s disease and ulcerativecolitis. Nat Clin Pract Gastroenterol Hepatol 3:390–407.

2. Hayden MS, Ghosh S (2004) Signaling to NF-�B. Genes Dev 18:2195–2224.3. Viemann D, et al. (2004) Transcriptional profiling of IKK2/NF-�B- and p38 MAP kinase-

dependent gene expression in TNF-�-stimulated primary human endothelial cells.Blood 103:3365–3373.

4. Karin M, Lin A (2002) NF-�B at the crossroads of life and death. Nat Immunol 3:221–227.5. Hacker H, Karin M (2006) Regulation and function of IKK and IKK-related kinases. Sci

STKE 2006, re13.6. Karin M, Yamamoto Y, Wang QM (2004) The IKK NF-�B system: A treasure trove for

drug development. Nat Rev Drug Discov 3:17–26.7. Broide DH, et al. (2005) Allergen-induced peribronchial fibrosis and mucus production

mediated by I�B kinase �-dependent genes in airway epithelium. Proc Natl Acad SciUSA 102:17723–17728.

8. Ruocco MG, et al. (2005) I�B kinase (IKK)� but not IKK� is a critical mediator ofosteoclast survival and is required for inflammation-induced bone loss. J Exp Med201:1677–1687.

9. McIntyre KW, et al. (2003) A highly selective inhibitor of I�B kinase, BMS-345541, blocksboth joint inflammation and destruction in collagen-induced arthritis in mice. ArthritisRheum 48:2652–2659.

10. Lawrance IC, et al. (2003) A murine model of chronic inflammation-induced intestinalfibrosis down-regulated by antisense NF-�B. Gastroenterology 125:1750–1761.

11. Neurath MF, Pettersson S, Meyer zum Buschenfelde KH, Strober W (1996) Localadministration of antisense phosphorothioate oligonucleotides to the p65 subunit ofNF-�B abrogates established experimental colitis in mice. Nat Med 2:998–1004.

12. MacMaster JF, et al. (2003) An inhibitor of I�B kinase, BMS-345541, blocks endothelialcell adhesion molecule expression and reduces the severity of dextran sulfate sodium-induced colitis in mice. Inflamm Res 52:508–511.

13. Greten FR, et al. (2004) IKK� links inflammation and tumorigenesis in a mouse modelof colitis-associated cancer. Cell 118:285–296.

14. Nenci A, et al. (2007) Epithelial NEMO links innate immunity to chronic intestinalinflammation. Nature 446:557–561.

15. Greten FR, et al. (2007) NF-�B is a negative regulator of IL-1� secretion as revealed bygenetic and pharmacological inhibition of IKK�. Cell 130:918–931.

16. Strnad J, Burke JR (2007) I�B kinase inhibitors for treating autoimmune and inflam-matory disorders: Potential and challenges. Trends Pharmacol Sci 28:142–148.

17. Egan LJ, et al. (2004) I�B-kinase�-dependent NF-�B activation provides radioprotectionto the intestinal epithelium. Proc Natl Acad Sci USA 101:2452–2457.

18. Chen LW, et al. (2003) The two faces of IKK and NF-�B inhibition: Prevention of systemicinflammation but increased local injury following intestinal ischemia-reperfusion. NatMed 9:575–581.

19. Egan LJ, et al. (2003) Nuclear factor-�B activation promotes restitution of woundedintestinal epithelial monolayers. Am J Physiol 285:C1028–C1035.

20. Nagashima K, et al. (2006) Rapid TNFR1-dependent lymphocyte depletion in vivo witha selective chemical inhibitor of IKK�. Blood 107:4266–4273.

21. Li X, et al. (2002) IKK�, IKK�, and NEMO/IKK� are each required for the NF-�B-mediatedinflammatory response program. J Biol Chem 277:45129–45140.

22. Wu F, Chakravarti S (2007) Differential expression of inflammatory and fibrogenicgenes and their regulation by NF-�B inhibition in a mouse model of chronic colitis.J Immunol 179:6988–7000.

23. Tanaka K, et al. (2007) Genetic evidence for a protective role for heat shock factor 1 andheat shock protein 70 against colitis. J Biol Chem 282:23240–23252.

24. Stephanou A, Latchman DS (1999) Transcriptional regulation of the heat shock proteingenes by STAT family transcription factors. Gene Expression 7:311–319.

25. el Marjou F, et al. (2004) Tissue-specific and inducible Cre-mediated recombination inthe gut epithelium. Genesis 39:186–193.

26. Li ZW, et al. (2003) IKK� is required for peripheral B cell survival and proliferation.J Immunol 170:4630–4637.

27. Kuhn R, et al. (1993) Interleukin-10-deficient mice develop chronic enterocolitis. Cell75:263–274.

28. Fort MM, et al. (2005) A synthetic TLR4 antagonist has antiinflammatory effects in twomurine models of inflammatory bowel disease. J Immunol 174:6416–6423.

29. Peterson RL, et al. (1998) Molecular effects of recombinant human interleukin-11 in theHLA-B27 rat model of inflammatory bowel disease. Lab Invest 78:1503–1512.

30. Qualls JE, Kaplan AM, van Rooijen N, Cohen DA (2006) Suppression of experimentalcolitis by intestinal mononuclear phagocytes. J Leukocyte Biol 80:802–815.

31. Sikora A, Grzesiuk E (2007) Heat shock response in gastrointestinal tract. J PhysiolPharmacol 58(Suppl 3):43–62.

32. Laubitz D, et al. (2006) Gut myoelectrical activity induces heat shock response inEscherichia coli and Caco-2 cells. Exp Physiol 91:867–875.

33. Rennick DM, Fort MM (2000) Lessons from genetically engineered animal models. XII.IL-10-deficient (IL-10�/�) mice and intestinal inflammation. Am J Physiol 278:G829–G833.

34. Karin M, Greten FR (2005) NF-�B: Linking inflammation and immunity to cancerdevelopment and progression. Nat Rev Immunol 5:749–759.

35. Madison BB, et al. (2002) Cis elements of the villin gene control expression in restricteddomains of the vertical (crypt) and horizontal (duodenum, cecum) axes of the intestine.J Biol Chem 277:33275–33283.

36. Katakura K, et al. (2005) Toll-like receptor 9-induced type I IFN protects mice fromexperimental colitis. J Clin Invest 115:695–702.

37. Hammer M, et al. (2006) Dual specificity phosphatase 1 (DUSP1) regulates a subset ofLPS-induced genes and protects mice from lethal endotoxin shock. J Exp Med 203:15–20.

Eckmann et al. PNAS � September 30, 2008 � vol. 105 � no. 39 � 15063

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