Mitogen-Activated Protein Kinase-Dependent Interleukin-1�Intracrine Signaling Is Modulated by YopP duringYersinia enterocolitica Infection

Rumu Bose, Josephine Thinwa, Paola Chaparro, Youmin Zhong, Santanu Bose, Guangming Zhong, and Peter H. Dube

The University of Texas Health Science Center at San Antonio, Department of Microbiology and Immunology, San Antonio, Texas, USA

Yersinia enterocolitica is a food-borne pathogen that preferentially infects the Peyer’s patches and mesenteric lymph nodes,causing an acute inflammatory reaction. Even though Y. enterocolitica induces a robust inflammatory response during infection,the bacterium has evolved a number of virulence factors to limit the extent of this response. We previously demonstrated thatinterleukin-1� (IL-1�) was critical for the induction of gut inflammation characteristic of Y. enterocolitica infection. More re-cently, the known actions of IL-1� are becoming more complex because IL-1� can function both as a proinflammatory cytokineand as a nuclear factor. In this study, we tested the ability of Y. enterocolitica to modulate intracellular IL-1�-dependent IL-8production in epithelial cells. Nuclear translocation of pre-IL-1� protein and IL-1�-dependent secretion of IL-8 into the culturesupernatant were increased during infection with a strain lacking the 70-kDa virulence plasmid compared to the case duringinfection with the wild type, suggesting that Yersinia outer proteins (Yops) might be involved in modulating intracellular IL-1�signaling. Infection of HeLa cells with a strain lacking the yopP gene resulted in increased nuclear translocation of pre-IL-1� andIL-1�-dependent secretion of IL-8 similar to what is observed with bacteria lacking the virulence plasmid. YopP is a proteinacetylase that inhibits mitogen-activated protein kinase (MAP kinase)- and NF-�B-dependent signal transduction pathways.Nuclear translocation of pre-IL-1� and IL-1�-dependent secretion of IL-8 in response to Yersinia enterocolitica infection weredependent on extracellular signal-regulated kinase (ERK) and p38 MAP kinase signaling but independent of NF-�B. These datasuggest that Y. enterocolitica inhibits intracellular pre-IL-1� signaling and subsequent proinflammatory responses through in-hibition of MAP kinase pathways.

There are three species of Yersinia pathogenic for humans, in-cluding the two enteric pathogens Y. enterocolitica and Y. pseu-

dotuberculosis as well as Y. pestis, the causative agent of plague (20).Y. enterocolitica and Y. pseudotuberculosis are both food-bornepathogens that infect the Peyer’s patches and mesenteric lymphnodes, causing a self-limiting infection (11, 12, 20). Initially, thebacteria attach to and invade M cells, which make up a specializedintestinal epithelium that overlays the Peyer’s patches (27, 31). Inrare cases, often in the context of immune compromise, systemicinfections involving most body systems can occur (12). Yersiniainfection is characterized by an acute inflammatory response thatis initiated by proinflammatory cytokines, leading to the recruit-ment and activation of neutrophils and macrophages (14–16, 21–23). Ultimately, a CD4� T-helper type 1 response clears the infec-tion (1–3).

Using animal models and cell culture, we and others demon-strated that interleukin-1 (IL-1) plays a critical role in initiatingthe inflammatory response to Y. enterocolitica infection (5, 6, 23).The IL-1 family consists of proinflammatory cytokines and in-cludes a number of molecules important for the host response toY. enterocolitica infection, such as IL-1�, IL-1�, and IL-18 (5–8,19, 23, 41). These cytokines are produced as preproteins that re-quire proteolytic cleavage to remove the propiece prior to secre-tion. IL-1 family members are differentially processed, with IL-1�and IL-18 being substrates of caspase-1 and the inflammasomeand IL-1� being cleaved by calpain (19, 41). Mature IL-1 familymembers are secreted from cells, and they subsequently act toinitiate inflammatory signaling on a variety of cell types. UnlikeIL-1� and IL-18, pre- and pro-IL-1� are biologically active, uti-lizing a nuclear localization sequence (NLS) at amino acids 79 to

86 to translocate from the cytoplasm to the nucleus, where IL-1�enhances the transcription of other proinflammatory cytokines,such as IL-8 (17, 38). Nuclear pre-IL-1� is known to interact withproteins associated with the transcriptional machinery, includingnecdin, GAL4, and histone acetyltransferase (13, 26, 37). It is nowhypothesized that the predominant role of IL-1� is as an intracel-lular signaling molecule. In addition to IL-1� being a nuclear fac-tor, translocation of IL-1� to the nucleus may serve as a means oflimiting inflammation during necrosis, when pro-IL-1� can func-tion as a danger-associated molecular pattern (DAMP) molecule.

Even though Yersinia infection leads to acute inflammation aspart of the host response, Y. enterocolitica has evolved numerousmechanisms to temper the host’s inflammatory response (20).Immune evasion molecules utilized by Yersinia are encoded onboth the chromosome and the 70-kDa virulence plasmid (pYv).Certain strains of Y. enterocolitica encode three distinct type threesecretion systems (TTSS), including chromosomal and flagellarTTSS, but the best-studied immune modulating mechanisms areassociated with the pYv-encoded TTSS and associated effectorproteins (18, 24, 40). TTSS allow Yersinia to directly secrete effec-

Received 2 August 2011 Returned for modification 27 August 2011Accepted 1 November 2011

Published ahead of print 14 November 2011

Editor: J. B. Bliska

Address correspondence to Peter H. Dube, dube@uthscsa.edu.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

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tor proteins from the bacteria directly into the cytoplasm of hostcells. The TTSS effector proteins known as Yops are enzymes thatmimic host proteins such as phosphatases, kinases, GTPase-activating proteins (GAPs), acetylases, and proteases that impacthost cell physiology by disrupting signal transduction pathwaysand the cytoskeleton (18). YopP (YopJ in Y. pestis and Y. pseudo-tuberculosis) is a protein acetylase that ultimately inhibits NF-�B,extracellular signal-regulated kinase (ERK), p38, and JunN-terminal protein kinase (JNK) signal transduction pathways byacetylating activating serine and threonine residues on the activat-ing kinases in these pathways (9, 33, 34). The action of YopP has avariety of consequences depending on the cell type being infected,but YopP can lead to the inhibition of proinflammatory cytokineproduction (tumor necrosis factor alpha [TNF-�]) and in macro-phages promotes apoptosis (4, 9, 32, 35). In mouse models of Y.enterocolitica infection, deletion of YopP has a measurable impacton virulence, but it is not an essential virulence factor in the highlymouse virulent serogroup 0::8 strains (36).

Infection of human epithelial cells with Y. enterocolitica leads tothe secretion of IL-8, and following infection in the mouse model,there is a rapid increase in the mouse IL-8 homologues KC andMip-1� (28). More recently, it was shown that during a Chla-mydia trachomatis infection, IL-8 production was dependent onpre-IL-1� intracellular signaling (17). Based on these observa-tions, we investigated the hypothesis that pre-IL-1� was respon-sible or partially responsible for the IL-8 observed after Y. entero-colitica infection of epithelial cells and that pre-IL-1� intracellularsignaling might be a target for Yersinia-mediated immune modu-lation.

MATERIALS AND METHODSBacteria. Yersinia enterocolitica strains used in this study are derivatives ofthe serogroup 0::8 strain 8081 and have been described previously (21,22). JB-580v is the virulent, wild-type (WT) strain, and JB-580c is anisogenic avirulent derivative of JB-580v lacking the pYv virulence plasmid.The yopP mutant has an in-frame deletion of yopP on the JB-580v back-ground. Bacteria were grown in LB broth containing 20 �g/ml nalidixicacid at 28°C overnight, diluted into fresh media, and cultured at 37°C for2 h prior to use.

Cell culture. HeLa cells were grown in Dulbecco’s modified Eagle’smedium (DMEM) supplemented with 10% heat-inactivated fetal bovineserum (FBS) at 37°C in a 5% CO2 atmosphere. Cells were cultured in24-well culture dishes on glass coverslips prior to analysis. Cells wereinfected with bacteria at a multiplicity of infection (MOI) of approxi-mately 5 for 30 min. Cells were then washed to remove nonadherentbacteria, fresh media containing 100 �g/ml gentamicin were added for 30min to kill extracellular bacteria, and then cells were maintained in mediawith 8 �g/ml gentamicin.

Plasmids and viruses. We have described the pre-IL-1�–red fluores-cent protein (RFP) construct previously (17). ERK 1/2, p38, and IL-1�short hairpin RNAs (shRNAs) and control shRNAs were purchased fromOriGene. For transfection, HeLa cells were transfected using Lipo-fectamine 2000 with 500 ng of pre-IL-1�–RFP and/or 500 ng of shRNAplasmids 24 or 48 h before use. Efficiency of the RNA interference (RNAi)was determined by reverse transcription-PCR (RT-PCR) (23). HeLa cellswere infected with adenovirus at an MOI of 6 for 48 h prior to use. Wehave described the I�B superrepressor-expressing virus and green fluo-rescent protein (GFP)-expressing viruses and their use previously (10).

Signal transduction inhibitors. Signal transduction inhibitors thatselectively block p38 mitogen-activated protein kinase (MAPK) (SB202190), MEK (U0126), c-RAF-1, and I�B kinase (IKK)–NF-�B (IKK-2inhibitor) were purchased from Calbiochem and dissolved in dimethylsulfoxide (DMSO). DMSO was used as a solvent control in all inhibitor

studies. Cells were treated with inhibitor SB-202190 (20 �M), U0126 (10�M), IKK-2 (100 ��), c-RAF-1 (9 nM), or DMSO for 2 h prior to infec-tion and maintained in the media for the course of the experiment. Cellswere monitored for cytotoxicity by lactate dehydrogenase (LDH) releaseassay, and the concentrations of signal transduction inhibitor used inthese studies did not induce any detectable signs of cytotoxicity.

Cell fractionation. HeLa cells were grown and treated as describedabove before being scrapped from the dish. Cells were washed once withice-cold phosphate-buffered saline (PBS) and collected at 2,600 rpm for 6min. Cells were resuspended in 450 �l buffer A (100 mM HEPES, pH 7.9,100 mM KCl, 0.5 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol [DTT],1 mM phenylmethylsulfonyl fluoride [PMSF]) and vortexed four timesfor 30 s. Extracts were collected by centrifugation 8,000 rpm for 8 min.The supernatant was the cytoplasmic fraction. The pellet was then resus-pended in 100 �l buffer C (200 mM HEPES, pH 7.9, 500 mM KCl, 5.0 mMEDTA, 1.0 mM EGTA, 1 mM DTT, 1 mM PMSF) by vortexing 3 times for30 s. This solution was collected by centrifugation 10,000 � g for 12 min at4°C. The supernatant was the nuclear fraction. Protein concentrations inthe fractions were estimated by Bradford assay, and the purity of thefractions was tested by Western blotting for tubulin and lamin to definethe cytoplasmic and nuclear fractions, respectively.

Pre-IL-1� nuclear localization and immunofluorescence. Endoge-nous pre-IL-1� was detected by indirect immunofluorescence. Briefly,HeLa cells were plated on coverslips and treated/infected as indicated. Atthe indicated time points, the cells were fixed in 2% paraformaldehyde for20 min prior to being placed in blocking/staining buffer (PBS, pH 7.4,containing 0.025% saponin, 1% bovine serum albumin [BSA], and 0.1%nonfat dry milk). IL-1� was detected using the mouse monoclonal anti-human IL-1� antibody clone MAB4145 from R&D Systems at 1:1,000 anda goat anti-mouse Alexa Fluor 568 secondary antibody at 1:2,000. Theanti-IL-1� antibody binds an epitope in proprotein corresponding to theregion that is processed to release the mature/secreted protein. Coverslipswere counterstained with Hoechst dye to visualize the nucleus. Stainingwas detected with a Zeiss Axioscope-2 microscope equipped with a digitalcamera and analyzed with the Axiovision suite of software. To quantifyIL-1� nuclear localization, 4 random fields (magnification, 40�)/cover-slip were imaged in the red and blue channels. The number of cells in thefield with nuclear localization of IL-1� was determined (red nucleus).Then, the total numbers of cells in the field (blue nuclei) were determined.The ratio of cytoplasmic to nuclear IL-1� was obtained and averaged foreach field. Each experiment was done in duplicate with at least three in-dependent repeats. Data are presented as the average � the standard de-viation.

ELISA. An enzyme-linked immunosorbent assay (ELISA) using aDuoSet ELISA kit from R&D Systems determined the levels of IL-8 se-creted into the culture supernatant as we have described previously (21).Extracellular IL-1� was assayed for using a DuoSet ELISA kit from R&DSystems. Cellular concentrations of IL-1� were determined using ELISAand expressed as pg cytokine/ml/�g total cellular protein as determinedby Bradford assay. Each experiment was done in triplicate with at least twoto four independent repeats. Data are presented as averages � standarddeviations. In each figure, the IL-8 ELISA is from the same experiment asthe corresponding IL-1� nuclear translocation data.

Statistical analysis. Data are presented as averages � standard devia-tions, and statistical significance was determined by two-tailed analysis ofvariance (ANOVA) with P values of �0.05 considered significant.

RESULTSTranslocation of pre-IL-1� to the nucleus after Y. enterocoliticainfection of HeLa cells. IL-1� is made as a proprotein containinga nuclear localization signal (NLS) located from amino acids 79 to86 in the propiece (Fig. 1A) (38). Infection of HeLa cells with Y.enterocolitica lacking the pYv virulence plasmid (JB-580c) or treat-ment with lipopolysaccharide (LPS) led to a significant (P � 0.05)increase in nuclear IL-1� relative to that in the nontreated cells or

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cells infected with wild-type Y. enterocolitica (JB-580v; Fig. 1B andC). At the time points tested, using ELISA, no extracellular IL-1�was detected in the cell culture supernatants in any of the experi-ments (not shown).

HeLa cells infected with JB-580v were most similar to the un-treated controls, suggesting that genes carried on the pYv viru-lence plasmid might be involved in modulating IL-1� nucleartranslocation (Fig. 1B and C). To evaluate the functional signifi-cance of nuclear IL-1�, we measured the secretion of IL-8 into theculture supernatant following infection with Y. enterocolitica (Fig.1D). Levels of IL-8 secretion following Y. enterocolitica infectionstrongly correlated with nuclear IL-1�; significantly more IL-8was secreted from cells infected with JB-580c or treated with LPSthan from nontreated cells or cells infected with JB-580v (Fig. 1D).Nontreated cells did not secrete detectable levels of IL-8, andthroughout the study, IL-1� was not detected in the cell superna-tant in any experimental condition tested (data not shown). Alto-gether, these data suggest that genes carried on the pYv virulenceplasmid significantly decrease the amount of IL-1� translocatingto the nucleus and the amount of IL-8 secreted into culture super-natant.

YopP inhibits IL-1� nuclear translocation and correspond-ing IL-8 secretion. The data presented in Fig. 1 strongly suggestthat genes carried on pYv modulate intracellular IL-1� signaling.pYv contains a number of genes that encode Yops, secreted viru-

lence factors which modulate the host immune response duringinfection (18). YopP is well known to inhibit proinflammatorycytokine production by inhibiting MAP kinase- and NF-�B-dependent pathways (34). To determine if the total IL-1� proteinconcentration changed after infection with Y. enterocolitica,ELISA was performed on HeLa cell extracts infected with variousY. enterocolitica strains. As shown in Fig. 2A, cells infected with astrain lacking pYv or yopP contained more IL-1�/�g cellular pro-tein than cells infected with the WT strain. HeLa cells infected witha Y. enterocolitica strain having an in-frame deletion of the yopPgene had significantly more IL-1� (P � 0.05) in the nucleus thanthe cells infected with the isogenic wild-type strain JB-580v (Fig.2B). Interestingly, the levels of nuclear IL-1� corresponding to theyopP mutant and the strain lacking the pYv virulence plasmid(JB-580c) were similar (Fig. 2B), suggesting that YopP modulatesnuclear IL-1� translocation. HeLa cells infected with JB-580c orthe yopP mutant secreted significantly more IL-8 into the culturesupernatant than cells infected with JB-580v (Fig. 2C). To test thecompartmentalization of IL-1� after infection by another means,we performed cellular fractionation and measured IL-1� concen-trations in the cytoplasmic and nuclear fractions. As shown in Fig.3A, relatively pure fractions were used in the analysis as deter-mined by �-tubulin for the cytoplasmic fraction and lamin A forthe nuclear fraction. Consistent with our immunofluorescence-based assay, cellular fractionation revealed significantly more

FIG 1 Inhibition of IL-1� nuclear translocation is dependent on the Pyv virulence plasmid. (A) Schematic of the pre-IL-1� protein showing the relative positionof the nuclear localization signal within the proprotein. (B) Representative indirect immunofluorescence images of pre-IL-1� nuclear translocation in HeLa cells4 h postinfection. IL-1� is in red and the nucleus is in blue. (C) Enumeration of nuclear IL-1� 4 and 22 h postinfection with wild-type (JB-580v) or plasmid-cured(JB-580c) Y. enterocolitica. Indirect immunofluorescence determined the percentage of nuclear IL-1� following infection. Data are averages � standarddeviations of two independent experiments done in duplicate (�, P � 0.05). (D) Concentrations of IL-8 secreted into the culture supernatant were determinedby ELISA 4 and 22 h postinfection with the indicated strains of Y. enterocolitica or controls. Data are averages � standard deviations of two independentexperiments done in triplicate (�, P � 0.05).

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(P � 0.0005) nuclear IL-1� when cells were treated with LPS orinfected with JB-580C or the yopP mutant (Fig. 3B). Likewise,the WT strain, JB-580v, had significantly more (P values of 0.01to �0.005) cytoplasmic IL-1� than cells infected with JB-580cor the yopP mutant or those treated with LPS (Fig. 3B). Alto-gether, these data strongly suggest that YopP modulates thenuclear translocation of IL-1� and the subsequent IL-1�-dependent production of IL-8.

IL-1� RNAi decreases IL-8 secretion following Y. enteroco-litica infection. Data presented in Fig. 2 and 3 could also be inter-preted to suggest that there is no correlation between IL-1� andIL-8 and that bacteria lacking Yops or YopP in particular justmake more IL-1� and IL-8. Therefore, to test if nuclear localiza-tion of IL-1� following Y. enterocolitica infection was correlated to

IL-8 secretion, we employed RNAi technology. Plasmids encodingshort hairpin RNAs (shRNAs) directed against the IL-1� tran-script were purchased from OriGene and screened by RT-PCR.As shown in Fig. 4A, HeLa cells transfected with an IL-1� overex-pression plasmid and shRNA for 24 h showed varying levels ofIL-1�-specific RNA inhibition relative to the control shRNA. Spe-cific shRNA 2 (Fig. 4A, lane 4) showed the best inhibition and wasused in all subsequent experiments. Treatment of HeLa cells withIL-1�-specific shRNA for 24 h prior to infection significantly (P �0.05) reduced the levels of IL-8 secreted into the culture superna-tant relative to cells treated with control shRNA (Fig. 4B). Longertreatment with shRNA (for 48 h) resulted in a slightly improvedinhibition with significantly (P � 0.0005 for all groups) reducedlevels of IL-1� and IL-8 compared to those in cells treated withcontrol shRNA (Fig. 5A and B). Using transient transfection ofIL-1�-specific shRNA, we were never able to completely removeIL-1� protein from the HeLa cells, but we could consistently re-duce it by two-thirds, which corresponded to a two-thirds de-crease in IL-8. Altogether, these data suggest that IL-8 productionfollowing Y. enterocolitica infection is partly dependent on intra-cellular IL-1�.

IL-1� nuclear translocation and IL-8 production is indepen-dent of NF-�B. Many proinflammatory cytokines are dependenton transcriptional activation by NF-�B. Indeed, the expression ofIL-1 is partially controlled by NF-�B, and NF-�B is often a targetof pathogen-mediated immune modulation; for example, Y. en-terocolitica uses YopP to inhibit NF-�B through the acetylation ofI�B (33, 34). To investigate the role of NF-�B in pre-IL-1� intra-cellular signaling, we first evaluated cellular responses to infectionwhen cells were pretreated with an IKK-2 inhibitor. BlockingNF-�B activation via chemical inhibitor had no impact on thelevels of nuclear IL-1�. As shown in Fig. 6A and B, cells treatedwith IKK-2 inhibitor and those treated with solvent had nearlyidentical percentages of nuclear IL-1� and concentrations of IL-8secreted into the culture supernatant at 6 h postinfection. Further,inhibitor treatment or solvent treatment had no impact on thepatterns of pro-IL-1� nuclear localization or IL-8 secretion. Forexample, irrespective of treatment with inhibitor, cells infectedwith JB-580c or the yopP mutant or treated with LPS still had morenuclear IL-1� and secreted more IL-8 into the supernatant thancells infected with JB-580v or untreated cells. To determine if the

FIG 2 YopP inhibits nuclear IL-1� and IL-8 secretion following Y. enterocolitica infection of HeLa cells. (A) HeLa cells were infected with the indicated strainsof Y. enterocolitica at an MOI of 5, and then the amount of IL-1�/�g cellular protein was determined 30 min, 4 h, and 6 h postinfection by ELISA. (B) HeLa cellswere infected with the indicated strains of Y. enterocolitica at an MOI of 5, and then the percentage of nuclear IL-1� was determined 0, 4, and 6 h postinfectionas described in Materials and Methods. NT, no treatment. (C) Concentrations of IL-8 secreted into the culture supernatant were determined by ELISA 0, 4, and6 h postinfection with the indicated strains of Y. enterocolitica or controls. Data are the averages � standard deviations of four independent experiments done intriplicate (�, P � 0.05; ���, P � 0.005).

FIG 3 Cellular fractionation revealed increased nuclear IL-1� following yopPmutant infection. (A) Cells were fractionated to cytoplasmic and nuclear frac-tions 6 h after infection with the indicated bacteria, and the quality of thefractionation was determined by Western blotting for cytoplasmic and nuclearmarkers (�-tubulin and lamin A, respectively). Lanes: 1, no treatment; 2, LPStreatment; 3, JB-580c; 4, JB-580v; 5, yopP mutant. (B) ELISA determined theconcentration of IL-1� in the cytoplasmic and nuclear fractions. Data arepresented as pg/ml IL-1� per �g protein and are the composites of two inde-pendent assays done in duplicate, reported as averages � standard deviations.�, P � 0.05; ���, P � 0.0005.

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IKK-2 inhibitor was functioning as expected, we treated differen-tiated U937 macrophages with IKK-2 inhibitor or solvent andthen stimulated them with LPS. As expected, macrophages treatedwith IKK-2 inhibitor produced significantly less TNF-�, an NF-�B-dependent cytokine, than the macrophages treated with sol-vent, indicating that inhibitor was working as expected (Fig. 6C).

This result was unexpected; therefore, to test the role of NF-�Bin pro-IL-1� signaling in another way, we utilized adenovirusesexpressing the I�B superrepressor or GFP as previously described(10). Cells were infected for 48 h with nonreplicating adenovirusesexpressing the I�B superrepressor or, as a control, GFP prior tobeing infected with Y. enterocolitica. Consistent with our resultsfrom the IKK-2 inhibitor experiments, expression of the I�B su-perrepressor or GFP had no impact on pre-IL-1�-dependent nu-

FIG 4 Silencing of IL-1� transcript significantly decreased IL-8 production following Y. enterocolitica infection of HeLa cells. (A) HeLa cells were transfected with0.5 �g of pDSred-IL-1� and shRNA specific to IL-1� or control shRNA for 24 h and then stimulated with LPS for 6 h before RNA was extracted for IL-1� andglyceraldehyde-3-phosphate dehydrogenase (GAPDH) RT-PCR to determine the optimal specific shRNA to be used in further experiments. Lanes: 1, transfectedwith pDSred-IL-1� and control shRNA; 2, not transfected; 3, pDSred-IL-1� and specific shRNA 1; 4, pDSred-IL-1� and specific shRNA 2; 5, pDSred-IL-1� andspecific shRNA 3; 6, pDSred-IL-1� and specific shRNA 4. Specific shRNA 2 was used in all subsequent experiments. (B) HeLa cells were transfected with specificshRNA 2 or control shRNA and treated as indicated for 6 h. ELISA 6 h postinfection with the indicated strains of Y. enterocolitica or controls determinedconcentrations of IL-8 secreted into the culture supernatant. Data are averages � standard deviations of two independent experiments done in triplicate, and alltreatment groups treated with shRNA were statistically significant compared to controls (P � 0.05).

FIG 5 Treatment of HeLa cells with IL-1�-specific shRNA led to reducedlevels of intracellular IL-1� and secreted IL-8. HeLa cells were transfected withIL-1�-specific shRNA 2 or control shRNA for 48 h and then infected with theindicated strain of Y. enterocolitica at an MOI of 5 for 6 h. (A) Concentration ofcellular IL-1�/�g protein after 6 h of infection. (B) Concentration of IL-8 inthe supernatant expressed per pg cellular IL-1� 6 h postinfection. Data are theaverages of two independent experiments. All data points are statistically sig-nificant (P � 0.0005) in comparisons of control shRNA and IL-1�-specificshRNA for a given infecting strain.

FIG 6 Chemical inhibition of the NF-�B pathway has little impact on nuclearIL-1� or IL-8 secretion. HeLa cells were treated with 100 �M IKK-2 inhibitoror solvent (DMSO) for 2 h prior to being treated as indicated. (A) The per-centage of nuclear IL-1� was determined 6 h postinfection. (B) Concentra-tions of IL-8 secreted into the culture supernatant were determined by ELISA6 h postinfection with the indicated strains of Y. enterocolitica or controls. (C)As a positive control, U937 macrophages were treated with 100 �M IKK-2inhibitor or DMSO and then stimulated with LPS. ELISA determined concen-trations of TNF-� secreted into the culture supernatant (��, P � 0.005). Nocytotoxicity was observed during these experiments. Data are averages (�standard deviations) of three independent experiments done in triplicate.

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clear localization or IL-8 secretion into the culture supernatant(Fig. 7A and B). To determine if the adenoviruses were function-ing as expected, we infected differentiated U937 macrophageswith adenoviruses expressing the I�B superrepressor or GFP andthen stimulated them with LPS. As expected, macrophages in-fected with adenoviruses expressing the I�B superrepressor pro-duced significantly less TNF-� than the macrophages infectedwith a virus expressing GFP, indicating that the viruses were work-ing as expected (Fig. 7C). Altogether, these data strongly suggestthat NF-�B plays a minimal role in pre-IL-1�-dependent intra-crine signaling events.

IL-1� nuclear translocation and IL-8 production are depen-dent on MAP kinase pathways. As shown in Fig. 2 and 3, modu-lation of nuclear localization and the associated secretion of IL-8are modulated by genes carried on pYv and more specificallyYopP. However, as shown in Fig. 6 and 7, pro-IL-1� signalingevents are independent of NF-�B in HeLa cells. In addition toNF-�B, YopP also inhibits signaling through MAP kinase path-ways. Previous work suggests that YopP/J works on the ERKand p38 pathways to block expression of TNF-� (9, 35). Up-stream of p38 and ERK is the c-Raf kinase. To test if c-Rafactivation leads to increased nuclear IL-1� and IL-8 secretion,HeLa cells were treated with a c-Raf inhibitor, and then pre-IL-1� nuclear localization and IL-8 production were evaluated.Inhibition of c-Raf led to a 50% decrease in both nuclear pre-IL-1� and IL-8 secretion into the culture supernatant (data notshown). These data suggest involvement of MAP kinase path-ways in IL-1� intracrine signaling.

c-Raf is not a target of YopP, and the effects of the c-Raf inhib-itor are likely indirect effects on the YopP targets ERK 1/2 and p38.To test the role of kinases downstream of c-Raf, we investigatedthe ERK 1/2 and p38 MAPK pathways. Involvement of p38 MAPkinase in pre-IL-1� signaling was tested through the use of the

selective p38 MAPK inhibitor SB-202190 or DMSO as a solventcontrol followed by infection with Y. enterocolitica. p38 MAPKinhibition potently and significantly (P, �0.05 to �0.005) inhib-ited pro-IL-1� nuclear translocation, reducing levels of nuclearIL-1� to baseline levels, whereas DMSO had no effect (Fig. 8A).Treatment of cells with SB-202190 also significantly (P � 0.005)reduced IL-8 production by approximately 50% compared to theDMSO controls (Fig. 8B). These data were in good agreementwith studies using shRNA directed against p38 MAPK, demon-strating significant reductions (P � 0.005 [control versus specificshRNA]) in both nuclear IL-1� and IL-8 (Fig. 8C and D). Thereduction in IL-8 following p38 MAPK shRNA or SB-202190treatment was similar in magnitude to what was observed in cellstreated with IL-1� shRNA, suggesting that an IL-1�–p38 MAPkinase-independent pathway contributes to the residual IL-8 ob-served during infection (Fig. 5 and 8). Altogether, these data sug-gest that p38 MAP kinase is an important signaling molecule lead-ing to pre-IL-1� nuclear translocation.

The involvement of the downstream p38 MAPK in pre-IL-1�nuclear translocation raised the possibility that the MEK 1/2 ki-nases might also be involved in the activation of pre-IL-1� signal-ing via ERK 1/2. To test the roles of MEK 1/2 in pre-IL-1� signal-ing, we treated HeLa cells with the MEK 1/2 inhibitor U0126 andmeasured IL-1� nuclear translocation and IL-8 production. Sim-ilar to what was observed in the p38 MAPK inhibitor studies,treatment of cells with U0126 led to significant (P, �0.05 to�0.005) decreases in the percentage of nuclear IL-1� and secre-tion of IL-8 following infection of these cells (Fig. 9A and B).Consistent with the p38 MAPK shRNA analysis, treating cells withshRNA directed at ERK 1/2 reduced the levels of nuclear IL-1�and IL-8 (Fig. 9C and D). Although MEK 1/2 inhibition did notreduce IL-1� nuclear localization to the levels observed with p38inhibition, these data suggest that both the ERK and p38 pathwayscontribute to IL-1� intracrine signaling.

FIG 7 Genetic inhibition of the NF-�B pathway has little impact on nuclearIL-1� or IL-8 secretion. HeLa cells were infected with adenoviruses expressingthe I�B superrepressor or GFP as a control for 48 h prior to being treated asindicated. (A) The percentage of nuclear IL-1� was determined 6 h postinfec-tion. (B) Concentrations of IL-8 secreted into the culture supernatant weredetermined by ELISA 6 h postinfection with the indicated strains of Y. entero-colitica or controls. (C) As a positive control, U937 macrophages were infectedwith adenoviruses expressing the I�B superrepressor or GFP prior to beingstimulated with LPS. Following LPS stimulation, the concentration of TNF-�was determined in the culture supernatant by ELISA. Data are averages �standard deviations of two independent experiments done in triplicate.

FIG 8 Chemical inhibition of p38 MAPK reduces nuclear IL-1� localizationand IL-8 secretion. HeLa cells were treated with 20 �M p38 inhibitor or solvent(DMSO) for 2 h prior to being treated as indicated, or they were transfectedwith p38 MAPK-specific shRNA. (A and C) The percentage of nuclear IL-1�was determined 6 h postinfection. (B and D) Concentrations of IL-8 secretedinto the culture supernatant were determined by ELISA 6 h postinfection withthe indicated strains of Y. enterocolitica or controls. Data are averages � stan-dard deviations of three independent experiments done in triplicate; all differ-ences in shRNA-treated cells (control versus specific) were statistically signif-icant (P � 0.05).

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Treatment of cells with both p38 MAPK and MEK 1/2 inhibi-tors or p38 and ERK 1/2 shRNA resulted in significant (P � 0.005)decreases in nuclear IL-1� and IL-8 production following infec-tion with Y. enterocolitica (Fig. 10A to D).

Our data suggest that YopP inhibits nuclear translocation ofIL-1� through modification of p38 MAPK and MEK 1/2. Thesedata would also predict that cells pretreated with p38 or MEK 1/2inhibitors would have more IL-1� in the cytoplasm and less in the

nucleus than cells treated with solvent controls when infected withthe yopP mutant. To test this directly, HeLa cells were pretreatedwith p38 MAPK inhibitor (SB-202190), MEK 1/2 inhibitor(U106), p38 plus MEK inhibitor, or DMSO as a solvent control for1 h. Cells were then treated with LPS or infected with JB-580v orthe yopP mutant for 6 h before being fractionated into cytoplasmicand nuclear fractions. As shown in Fig. 11A, there was significantly(P � 0.005) less IL-1� in both fractions when cells were treatedwith inhibitors, as inhibition of these signaling pathways does de-crease the overall levels of IL-1�. More importantly, there was anincrease in the nuclear-to-cytoplasmic IL-1� ratio when cells weretreated with p38 or MEK 1/2 inhibitors and then infected with theyopP mutant, suggesting an accumulation of cytoplasmic IL-1�.

Altogether, these data suggest that MAPK signaling cascadesare involved in IL-1� intracrine signaling, leading to subsequentIL-8 secretion, and that YopP action impacts MAPK signaling topartially modulate these intracrine signaling events (Fig. 8, 9, 10,and 11).

DISCUSSION

Yersinia enterocolitica infection causes self-limiting disease char-acterized by a robust inflammatory response in the Peyer’s patchesand mesenteric lymph nodes (14). Interestingly, the extensivenumber of virulence factors that Y. enterocolitica uses to modulatethe host’s immune response likely mutes the observed inflamma-tory response. Early during infection, members of the IL-1 family,including IL-1�, IL-1�, and IL-18, are important mediators of thehost response (5, 6, 8, 23). Previously, we showed that following Y.enterocolitica infection of the mouse, IL-1� is critical for the gen-eration of gut inflammation (23). Moreover, a number of otherstudies using the mouse model have highlighted the role of IL-1family members in the control of Y. enterocolitica infection (5, 6,8). However, these studies predominantly examined the secretedforms of these proteins, and while IL-1� and IL-18 function as

FIG 9 Chemical inhibition of MEK 1/2 reduces nuclear IL-1� localization and IL-8 secretion. HeLa cells were treated with 10 �M MEK 1/2 inhibitor or solvent(DMSO) for 2 h prior to being treated as indicated, or they were transfected with ERK 1/2 MAPK-specific shRNA. (A and C) The percentage of nuclear IL-1� wasdetermined 6 h postinfection. (B and D) Concentrations of IL-8 secreted into the culture supernatant were determined by ELISA 6 h postinfection with theindicated strains of Y. enterocolitica or controls. Data are averages � standard deviations of three independent experiments done in triplicate (�, P � 0.05; ���,P � 0.0005); all differences in shRNA-treated cells (control versus specific) were statistically significant (P � 0.05).

FIG 10 Chemical inhibition of p38 and MEK 1/2 pathways reduces nuclearIL-1� localization and IL-8 secretion. HeLa cells were treated with 10 �MU0126 plus 20 �M SB-202190, or they were transfected with p38 and ERK 1/2MAPK-specific shRNA and were then infected with the indicated strains of Y.enterocolitica. (A and C) The percentage of nuclear IL-1� 6 h postinfection. (Band D) The concentration of IL-8 secreted into the culture supernatant wasdetermined by ELISA 6 h postinfection. Data are averages � standard devia-tions of two independent experiments done in triplicate. All comparisons be-tween cells treated with inhibitors and DMSO controls were significantly dif-ferent (P � 0.005), and all differences in shRNA-treated cells (control versusspecific) were statistically significant (P � 0.05).

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secreted cytokines, IL-1� has a variety of other roles during thehost response.

IL-1� has a complex biology, and currently it is accepted thatthe major role of this protein is as an intracellular signaling mol-ecule and as a DAMP. As diagramed in Fig. 1A, the propiece ofIL-1� contains a nuclear localization signal, and Werman et al.demonstrated that this piece of the protein was sufficient to trans-locate heterologous proteins from the cytoplasm to the nucleus(37). Within the nucleus, IL-1� interacts with the transcriptionalmachinery (necdin and histone acetyltransferase) (13, 26). Nu-clear IL-1� can facilitate the expression of PAI-1 in endothelialcells (29, 30), IL-8 and IL-6 in epithelial cells (17), and presumablyother proinflammatory cytokines. The full repertoire of IL-1�-elicited intracrine responses is currently unknown.

IL-1� intracrine signaling is an important part of the host re-sponse to infection, leading to the production of the potent neu-trophil chemoattractant IL-8. Cheng and colleagues previouslydemonstrated the ability of IL-1� intracrine responses to lead toincreased expression of IL-6 and IL-8 during infection of HeLacells with the intracellular pathogen Chlamydia trachomatis (17).Indeed, the work presented in this study is complementary towhat was observed with Chlamydia and illustrates how Yersiniamodulates this aspect of the immune response. Interestingly, fol-lowing the initial invasion of epithelial cells, Y. enterocolitica ispredominantly an extracellular pathogen, making neutrophilsprominent cells in the host response to infection. In this report, wedemonstrate that YopP tempers intracrine IL-1� signaling andIL-8 production during a wild-type Y. enterocolitica infection. Wepreviously reported that infection of mice with a Y. enterocoliticarovA mutant, which does not induce IL-1� expression, also leadsto a dramatic reduction in the levels of neutrophil-mediated in-flammation that may also be linked to reductions in nuclear IL-1�and the expression of the IL-1�-dependent cytokines KC, Mip-1,and IL-6 (23, 25).

The first host cells encountered by Y. enterocolitica are intesti-nal epithelial cells, and presumably epithelial cells are the initialsites of Yop-mediated immune evasion. The binding to and inva-sion of epithelial cells by Y. enterocolitica can trigger IL-8 produc-tion in an invasin-dependent manner (28). Although we did notidentify the pathogen-associated trigger of IL-1� nuclear localiza-

tion and IL-8 production, based on previous work (27), it wouldbe reasonable to speculate that invasin-mediated integrin signal-ing is involved.

Epithelial cells are an important part of the innate immunesystem, and as such, they are capable of responding rapidly tomicrobial insult. In fact, epithelial cells play a central role in thepathogenesis of many gut pathogens, such as Shigella, Salmonella,and Yersinia species. The attachment to and invasion of epithelialcells by Y. enterocolitica likely represent the first opportunity forthe host to detect and respond to infection, and epithelial cells alsorepresent an initial site of pathogen-mediated innate immunemodulation. Several studies have shown that the TTSS effectorprotein YopP/J can negatively regulate proinflammatory cytokineexpression and impact cell survival in a variety of cell types (4, 35).YopP/J impacts many of the signaling pathways leading to proin-flammatory cytokine production, including NF-�B, ERK 1/2, p38,and JNK by acetylating the activation serine or threonine on theactivating or terminal kinases in these pathways (34). This conser-vation of biochemical action allows the virulence factor to inacti-vate a variety of signaling pathways at once or to block signaling insituations in which different cell types use pathways preferentially.Interestingly, in epithelial cells, signaling through p38 and ERK1/2 seems to be responsible for pro-IL-1�-dependent IL-8 pro-duction, whereas NF-�B does not appear to play a role in thisprocess. The fact that inhibition of both ERK 1/2 and p38 MAPKled to partial reductions in IL-8 and nuclear IL-1� suggests thatboth pathways, along with yet to be identified pathways suggestedby the residual IL-1� and IL-8 present following MAPK inhibi-tion, may be involved in this response. YopP-mediated inhibitionof p38 might function partially through posttranslational mecha-nisms, since p38 can be important for cytokine mRNA stabiliza-tion (39), but this remains to be tested during Y. enterocoliticainfection.

The mechanisms underlying the ability of YopP to interferewith IL-1� nuclear translocation are unknown. However, it hasbeen reported that IL-1� interacts with histone acetyltransferasesin the nucleus (26), and YopP is an acetylase as well, raising thepossibility of a direct interaction. Another possibility that is morelikely is that MAPKs phosphorylate a protein or proteins that pro-mote IL-1� nuclear translocation. Further investigation will illu-

FIG 11 IL-1� concentration increases in the cytoplasmic fraction of cells treated with MAPK inhibitors. HeLa cells were treated with the indicated inhibitors andthen treated with LPS or infected with JB-580v or the yopP mutant. After 6 h of infection, cells were fractionated into cytoplasmic and nuclear fractions. (A) ELISAdetermined the concentration of IL-1� in the indicated fraction. Data are presented as pg/ml IL-1�/�g protein. The ratio of nuclear to cytoplasmic IL-1� ispresented above the respective treatment/infection group. Drug treatment for each group is indicated. (B) Representative examples of cellular fractionation arepresented Lanes: 1, no treatment; 2, LPS treatment; 3, JB-580v infection; 4, yopP mutant infection. Data are presented as averages � standard deviations of twoindependent experiments done in duplicate (A) or representative examples (B).

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minate the mechanisms underlying control of intracrine signalingfollowing Yersinia infection.

Altogether, these data expand our understanding of the signal-ing pathways and effector molecules impacted by YopP. More-over, these data give insight into possible mechanisms of IL-1�intracrine signaling during the response to infectious insult. Fur-ther investigations will determine how IL-1� intracrine signalingis triggered in response to Y. enterocolitica infection and how YopPmodulates these responses to maintain pre-IL-1� in the cyto-plasm.

ACKNOWLEDGMENTS

The National Institutes of Health, through grants AI067716 and AI060789awarded to P.H.D. and AI083387 awarded to S.B. supported this work.

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