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Originally published In Press as doi:10.1074/jbc.M601988200 on July 13, 2006

J. Biol. Chem., Vol. 281, Issue 36, 26112-26120, September 8, 2006
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Suppression of NF-{kappa}B Activation by Entamoeba histolytica in Intestinal Epithelial Cells Is Mediated by Heat Shock Protein 27*

Srinivas J. Kammanadiminti1 and Kris Chadee2

From the Department of Microbiology and Infectious Diseases, University of Calgary, Calgary, Alberta T2N 4N1, Canada

Received for publication, March 2, 2006 , and in revised form, July 12, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Little is known about the pathogenesis of Entamoeba histolytica and how epithelial cells respond to the parasite. Herein, we characterized the interactions between E. histolytica and colonic epithelial cells and the role macrophages play in modulating epithelial cell responses. The human colonic epithelial cell lines Caco-2 and T84 were grown either as monoculture or co-cultured in transwell plates with differentiated human THP-1 macrophages for 24 h before stimulation with soluble amebic proteins (SAP). In naive epithelial cells, prolonged stimulation with SAP reduced the levels of heat shock protein (Hsp) 27 and 72. However in THP-1 conditioned intestinal epithelial cells SAP enhanced Hsp27 and Hsp72, which was dependent on the activation of ERK MAP kinase. Hsp synthesis induced by SAP conferred protection against oxidative and apoptotic injuries. Treatment with SAP inhibited NF-{kappa}B activation induced by interleukin-1beta; specifically, the NF-{kappa}B-DNA binding, nuclear translocation of p65 subunit, and phosphorylation of I{kappa}B-{alpha} were reduced. Gene silencing by small interfering RNA confirmed the role of Hsp27 in suppressing NF-{kappa}B activation at I{kappa}B kinase (IKK) level. By co-immunoprecipitation studies, we found that Hsp27 interacts with IKK-{alpha} and IKK-beta, and this association was increased in SAP-treated conditioned epithelial cells. Overexpression of wild type Hsp27 amplified the effects of SAP, whereas a phosphorylation-deficient mutant of Hsp27 abrogated SAP-induced NF-{kappa}B inhibition. In conditioned epithelial cells, Hsp27 was phosphorylated at serine 15 after prolonged exposure to SAP. This mechanism may explain the absence of colonic inflammation seen in the majority of individuals infected with E. histolytica.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The intestinal protozoan parasite Entamoeba histolytica affects 50 million people worldwide, causing 100,000 deaths per year (1). Intestinal amebiasis is characterized by colitis and severe dysentery. Despite our knowledge of the role of several pathogen and host factors in causing colonic inflammation, the cell-specific response to amebic infection is poorly understood. Moreover, the majority of research done to unravel the mechanism of amebic colitis has been focused on proinflammatory responses (2, 3) by epithelial cells and a role of NF-{kappa}B (4) and chemokines such as IL-83 (5) as triggering events for inflammation. However, it is noteworthy that only 10% of E. histolytica-infected individuals show symptoms of intestinal inflammation (6), and none of the studies addressed the question of why only a minority of infected individuals develops amebic colitis.

Epithelial cells are the first layer of host defense and have been shown to be the effector cells capable of secreting several mediators in response to pathogens (7, 8). Epithelial cells in vivo do not respond in isolation but act in concert with several immune and nonimmune cells present in the lamina propria. To simulate this, several in vitro studies were carried out to assess the epithelial cell responses in the presence of immune cells such as leukocytes and lymphocytes. Under these conditions, a differential response was observed in epithelial cell lines exposed to various pathogenic and nonpathogenic bacteria (9, 10). However, the effect of co-culturing with macrophages on epithelial cell responses has not been well characterized.

The universal response to stress has been the induction of a group of highly conserved family of proteins called heat shock proteins (Hsp) and is commonly referred to as heat shock response or stress response. Several pathogens and their products have been shown to induce various Hsp in different cell types including intestinal epithelial cells (11–13). Hsp serve to protect the cells against several insults such as thermal, toxic, or apoptotic stimuli (12, 14–16). Epithelial cell induction of Hsp in response to various pathogens such as E. coli, and toxins such as lipopolysaccharide and superantigen have been reported (10, 12, 14). It has also been shown that epithelial Hsp expression is regulated by cytokines and immune cells such as lymphocytes (13). One emerging concept is that stress response counters the inflammatory response mediated by NF-{kappa}B, helping to reduce inflammation and promote healing of damaged tissues (12, 17–20). Because proinflammatory cytokines secreted by immune cells have been shown to influence epithelial cell responses (16, 21) and macrophages are a major source of these cytokines, we studied the effect of macrophages on epithelial cell response toward E. histolytica. We hypothesized that amebae might be eliciting a protective response whereby inflammation is suppressed in the majority of infected individuals. Thus, we sought to study the stress response induced by ameba components in naïve and macrophage-conditioned colonic epithelial cells and made several interesting and novel observations. For the first time, we showed that macrophage conditioning primes epithelial cells for an augmented Hsp expression in response to amebic components. We identified Hsp27 as the key mediator suppressing NF-{kappa}B activation by virtue of its association with I{kappa}B kinase (IKK) complex in intestinal epithelial cells (IEC). We conclude that this could be one of the mechanisms by which colonic inflammation is suppressed in the majority of E. histolytica-infected individuals and that the lack of such protective responses in a susceptible individual could lead to the symptoms associated with amebic colitis.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines, Amebic Components, and Reagents—Human colonic adenocarcinomal cell lines T84 and Caco-2 from ATCC (Manassas, VA) were used and maintained in Dulbecco's modified Eagle's medium-Ham's F-12 medium and minimal essential medium, respectively, supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin sulfate, and 20 mM HEPES (Sigma). Human THP-1 macrophages were maintained at 37 °C and 5% CO2 in complete RPMI 1640 (Invitrogen) supplemented with 10% heat-inactivated fetal calf serum (Hyclone Laboratories). To obtain adherent macrophages, 2 x 106 cells/well in 6-well culture plates were allowed to differentiate in the presence of 10 nM phorbol 12-myristate 13-acetate (Sigma) for 3 days. The macrophages were washed and made quiescent by incubation in complete RPMI 1640 medium for 24 h prior to co-culturing. Soluble amebic proteins (SAP) were prepared by three cycles of freeze-thaw lysis of log phase E. histolytica virulent strain HM1:IMSS and quantified by the BCA assay (Pierce). E. histolytica secretory components were prepared as described previously (5). Fas ligand and Caspase 3 antibody are from Oncogene. Antibody against NF-B p65 and MAP kinases, double-stranded NF-{kappa}B oligonucleotide (sc-2505), and all of the siRNA reagents are from Santa Cruz Biotech. Anti-IKK antibodies are from Cell Signaling Technology. Antibodies against human Hsp27, Hsp72, Hsp60, Hsp90, HSF-1 phospho-Hsp27 (Ser15), phospho-Hsp27 (Ser72), and hamster Hsp27 (Hsp25) are from Stressgen (Victoria, Canada). For immunoprecipitation, agarose-conjugated goat antibodies from Santa Cruz Biotech were employed: Hsp27 (sc-1048), Hsp60 (sc-1722), Hsp72 (sc-1060), and Hsp90 (sc-1055). For overexpression studies, hamster Hsp25 was used. PD98059 was obtained from Calbiochem. All other chemicals are from Sigma. The inhibitors galactose, E-64, and cycloheximide were used at slightly higher concentrations than those that have been previously demonstrated to have their intended effects (22–24).

Co-culturing of Epithelial Cells with Macrophages—T84 or Caco-2 cells between 10 and 30 passages grown in either regular or transwell plates for 7–10 days to achieve confluency were used. For siRNA experiments, subconfluent (40–50%) Caco-2 cells grown for 2–3 days were used. Human monocyte-like cell line THP-1 was differentiated with phorbol 12-myristate 13-acetate for 3 days and quiescence for 24 h before use. Transwells with epithelial cells were kept in culture plates containing 2 x 106 macrophages for 24 h (30h for siRNA studies), removed from co-culture, and immediately used for experiments. Epithelial cells were kept under low serum condition (5% fetal bovine serum) during co-culturing and subsequent treatments and without antibiotics for siRNA studies.

Western Blot—Cellular extracts from amebic protein-treated epithelial cells were prepared by scraping into the sample buffer containing SDS and mercaptoethanol and boiled for 10 min, and equal volumes were separated in 12% SDS-polyacrylamide gels and transferred onto nitrocellulose membrane (Bio-Rad). For nonreducing gel run, sample buffer without mercaptoethanol was used. The membranes were blocked in 3.5% skim milk-TBS-T (20 mM Tris-HCl, pH 7.5, 500 mM NaCl. 0.1% Tween 20) at 4 °C overnight, incubated with primary antibodies in 1% skim milk-TBS-T at 4 °C overnight, washed three times with TBS-T, and incubated with horseradish peroxidase-conjugated secondary antibody in skim milk-TBST overnight at 4 °C. After three washes each in TBS-T and TBS-3T, the blot was developed with the enhanced chemiluminescence system (Amersham Biosciences) according to the manufacturer's instructions.

Electrophoretic Mobility Shift Assay—Nuclear extracts were collected using the NE-PER kit (Pierce) and protein quantified by BCA assay. Annealed double-stranded heat shock element oligonucleotide–107 to –83 of the human Hsp70 gene (5'-GAT CTC GGC TGG AAT ATT CCC GAC CTG GCA GCC GA-3') (Sigma Genosys) or NF-{kappa}B consensus oligonucleotide (Santa Cruz Biotechnology) was labeled with [32P]ATP, using T4 polynucleotide kinase (Invitrogen). Unlabeled nucleotides were removed using Sephadex G-25 columns. The binding reaction consists of a 20-liter total volume of 0.5 ng of DNA probe, 5 g of nuclear extract, 1 g of poly(dI-dC) in the binding buffer (12 mM HEPES, 60 mM KCl, 4 mM MgCl2, 1 mM EDTA, 1 mM of dithiothreitol, and 12% glycerol, pH 8.0), and incubation for 30 min at room temperature. DNA-protein complexes were resolved by electrophoresis on 6% polyacrylamide gels at 4 °C in TBE buffer (90 mM Tris borate, 2 mM EDTA). The gels were subsequently dried and autoradiographed with intensifying screens at –70 °C.

Neutral Red Assay—Neutral red (Sigma) was reconstituted in serum-free medium and added to cells at 1.14 mM concentration. After 2 h of incubation, the medium was removed, and the cells were washed twice with phosphate-buffered saline; finally, the incorporated neutral red was released from the cells by incubation for 15 min at room temperature in the presence of 2 ml of the extraction buffer containing acetic acid (1%, v/v) and ethanol (50%, v/v). To measure the dye taken up, the cell lysis products were centrifuged, and the supernatants were spectrophotometrically measured at 540 nm.

Co-immunoprecipitation—Cells were lysed in 1% CHAPS buffer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl with 1% CHAPS) with protease inhibitor mixture (Roche Applied Science). 500 µg of cell extracts were incubated overnight with 5 µg of agarose-conjugated anti-Hsp antibodies (Santa Cruz Biotechnology). Precipitate was washed thrice with lysis buffer, dissolved in 2x Laemmli buffer, boiled, and separated by SDS-PAGE, transferred to nitrocellulose membrane, and detected by Western blot analysis using ECL (Amersham Biosciences).


Figure 1
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  		FIGURE 1.
Differential regulation of Hsp in naive and macrophage-conditioned epithelial cells. A, expression of Hsp in response to amebic lysate in naive epithelial cells. 100 µg/ml of SAP were added to naive Caco-2 cells, and cell lysate was collected at different time points and checked for the Hsp expression by Western blot as described under "Experimental Procedures." B, Caco-2 cells were co-cultured with differentiated THP-1 cells for 24 h and immediately stimulated with 100 µg/ml of SAP for different time periods, and equal volumes of cell extracts in Laemmli sample buffer were subjected to SDS-PAGE and Western blot. A representative blot from three independent experiments is shown. (***, p < 0.001; **, p < 0.01). The bottom panel shows representative blots from T84 cells. C, Caco-2 cells were co-cultured with THP-1 as described and stimulated with 100 µg/ml of SAP or ameba secretory components for 12 h and checked for Hsp27 expression. (**, p < 0.01; *, p < 0.05).

 
RNA Interference—Caco-2 cells were transected with siRNA against HSF-1 (sc-35611), Hsp27 (sc-29350), Hsp70 (sc-29352), or control siRNA (sc-37007) from Santa Cruz Biotechnology as per the manufacturer's protocol. Briefly, subconfluent (40–50%) Caco-2 cells were transfected using siRNA transfection reagent for 30 h during the co-culture at a siRNA concentration of 40 nM. The cells were immediately used for SAP and subsequent treatments.

Overexpression of Wild Type and Phosphorylation-deficient Mutant Hsp27 in M-IEC—Plasmids (pcDNA3.1) encoding wild type hamster Hsp25 and phosphorylation deficient-mutant Hsp25 (AA) in which Ser15 and Ser90 are replaced by alanine were kindly provided by Dr. T. Tomako (McGill University, Montreal, Canada). Caco-2 cells were transfected with empty plasmid (vector), wild type, and AA by FuGENE reagent (Promega) overnight according to the manufacturer's instructions. A transfection efficiency of 50–60% was observed using a green fluorescent protein control. The cells were recovered for 24 h while simultaneously co-cultured with differentiated THP-1, and then at the end of 24 h, conditioned IEC was treated with SAP and IL-1beta as described before.


Figure 2
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  		FIGURE 2.
Macrophage secretions prime IEC for Hsp induction. Caco-2 cells were treated with 24 h macrophage culture medium for 12 h before stimulating with SAP for different time points and checked for Hsp72 expression by Western blot. Representative blots from two experiments are shown (**, p < 0.01; *, p < 0.05).

 
In Vitro Kinase Assay—Cells were incubated with cell lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 25 mM beta-glycerophosphate, 2 mM EDTA, 2 mM pyrophosphate, 1 mM orthovanadate, and 1% Triton X-100, 1 mM dithiothreitol, 1 mM NaF with protease inhibitors) followed by the addition of anti-IKK-{alpha} antibody. Following overnight end-to-end rotation of tubes at 4 °C, immunoprecipitates were washed three times with lysis buffer and once with kinase buffer (20 mM Tris, pH 7.5, 1 mM MnCl2, 10 mM MgCl2, 20 mM beta-glycerophosphate, 0.1 mM sodium orthovanadate, 2 mM NaF, and 1 mM dithiothreitol). Immunoprecipitates were finally resuspended in 20.0 µlof kinase buffer containing 5 µCi of [{gamma}-32P]ATP and incubated at 30 °C for 30 min. 1 µg of glutathione S-transferase-I{kappa}B{alpha} (Santa Cruz Biotechnology) was used as substrate. The reaction was terminated by boiling with SDS sample buffer for 5 min. Finally, the protein was resolved on 10% SDS-PAGE, the gel was dried, and the radioactive bands were visualized by autoradiography. The cell lysates were also checked for IKK-{alpha} expression for normalization.


Figure 3
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  		FIGURE 3.
Ameba-induced Hsp expression in macrophage-primed IEC is dependent on ERK MAP kinase. A, M-IEC were pretreated with ERK MAP kinase inhibitor PD98059 at different concentrations for 2 h and then stimulated with SAP (100 µg/ml) for 12 h (***, p < 0.001). B, M-IEC treated with SAP for different time points and cell extracts probed with antibody against ERK MAP kinase. The blot shown is representative of three independent experiments. Ctl, control.

 
Statistical Analysis—The blots were scanned, and the densitometric values were obtained by using the Image J program. Statistical analysis to check significance was done with Student's t test using Prism software. The graphs plotted were from two to three independent experiments, and the error bars in all of the graphs represent the means ± S.D.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Differential Induction of Hsp27 and Hsp72 in Naive and Macrophage-conditioned Epithelial Cells—Because the induction of Hsp is a universal stress response against various insults and has been widely shown to have an anti-inflammatory effect, we checked the expression of Hsp representing four families. Although SAP treatment suppressed Hsp27 and Hsp72 in naïve IEC (Fig. 1A), the same two Hsp were significantly overexpressed in macrophage-conditioned Caco-2 and T84 colonic epithelial cells (Fig. 1B). Expression levels of Hsp60 and 90 remained unaltered in both naïve and conditioned IEC (not shown). Surprisingly, even though both secretory components and SAP elicited this stress response, SAP was consistently found to be twice as potent as secretory components (Fig. 1C) and was therefore used for all further studies. It should be noted that co-culturing alone did not affect the basal Hsp expression significantly (Fig. 1B). Also, treatment of IEC with 24 h THP medium was sufficient to prime epithelial cells for ameba-induced Hsp72 induction (Fig. 2) without the need for co-culturing. This confirms that macrophage secretions can alter epithelial cell responses to pathogens.


Figure 4
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  		FIGURE 4.
SAP induced Hsp72 induction is independent of Gal-lectin and cysteine protease. SAP was treated with 150 mM of D-galactose or 100 µM of E-64 for 1 h or boiled at 100 °C for 5 min before adding to the M-IEC for 12 h. Western blot was done as described under "Experimental Procedures." NS, not significant (**, p < 0.01).

 
Ameba-induced Hsp Expression Is ERK MAP Kinase-dependent—MAPK play important roles in regulating both inflammatory and stress responses (12) and Hsp have been shown to be regulated by different MAPK in intestinal epithelial cells (16, 25). Hence, we checked whether ameba-induced expression of Hsp27 and 72 are regulated by MAPK. As shown in Fig. 3A, when the conditioned epithelial cells was pretreated with PD98059, an ERK MAPK inhibitor, SAP-induced Hsp expression was significantly inhibited. Correspondingly we observed the activation of ERK MAPK by amebic proteins as early as 30 min (Fig. 3B).


Figure 5
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  		FIGURE 5.
Amebic protein induced Hsp72 expression is mediated by HSF-1. A, M-IEC were treated with 25 µM of quercetin (QC) or cycloheximide (Chx) for 2 h before stimulating with SAP for 12 h (**, p < 0.01). B, M-IEC transfected with different concentrations of siRNA against HSF-1 for 30 h as described under "Experimental Procedures." Cell lysates were subjected to SDS-PAGE, and Western blot was done with HSF-1 antibody. C, M-IEC cells were transfected or not with 40 nM HSF-1 siRNA for 30 h before treating with SAP for 12 h and checked for Hsp72 expression by Western blot as described. D, M-IEC was treated with SAP for different time points and cell extract collected and ran in a nonreducing gel and Western blot done with HSF-1 antibody as described. The arrows indicate activated dimeric and trimeric forms of HSF-1. E, M-IEC was incubated or not with quercetin (25 µM) for 2 h prior to treatment with SAP (100 µg/ml) for 3 h. 10.0 µg of nuclear extracts subjected to electrophoretic mobility shift assay as described. The blots shown are one of two to three replicates.

 
E. histolytica Induction of Hsp72 Is Independent of Gal-lectin and Cysteine Proteinase—The three well recognized virulent factors in E. histolytica are the surface adhesin Gal/GalNac lectin, cysteine proteinases, and amebapore. The pathogenic effects of the Gal-lectin and cysteine proteinases have been extensively documented (26), and it was of interest to determine whether the up-regulation of Hsp72 is mediated by these virulent factors. As shown in Fig. 4, pretreatment of SAP with either galactose or E-64 failed to inhibit Hsp72 expression, indicating that Hsp induction by SAP is independent of Gal-lectin and cysteine proteinases. In contrast, significant abrogation of Hsp72 induction was noted by boiled SAP, suggesting that a yet to be characterized protein moiety might be responsible for this effect.

Amebic Components Induce Hsp72 via HSF-1—We then studied the mechanism of up-regulation of Hsp72 by amebic components. Quercetin is a flavanoid compound and a known inhibitor of Hsp synthesis by inhibiting HSF-1 (27, 28), and it was of interest to determine whether it could inhibit SAP-mediated Hsp72 induction. As shown in Fig. 5A, quercetin (25 µM) significantly inhibited Hsp72 expression. Quercetins at high concentrations are known to inhibit protein synthesis. Thus, to confirm that quercetin inhibits SAP induced Hsp72 expression by inhibiting HSF-1 and not by inhibiting Hsp protein synthesis, we treated the cells with equimolar concentrations of cycloheximide, an inhibitor of protein synthesis. Under these conditions cycloheximide treatment failed to inhibit SAP induced Hsp72 expression, confirming that amebic components indeed induce Hsp synthesis by activating HSF-1. To conclusively prove the role of HSF-1 in ameba-induced Hsp synthesis, we silenced the HSF-1 gene by siRNA (Fig. 5B) and observed inhibition of Hsp72 expression by SAP in macrophage-treated epithelial cells (Fig. 5C). Consistent with its role in regulating Hsp72 expression, HSF-1 was activated by amebic proteins as shown by Western blot analysis (Fig. 5D). We found that amebic components were able to induce trimerization of HSF-1 in M-IEC at a time point of 3 h. There was a corresponding increase in the DNA binding activity of HSF-1, which was inhibited by pretreatment with quercetin (Fig. 5E). These studies confirm that amebic components activate HSF-,1 which in turn induces Hsp gene expression.

Heat Shock Response by Amebic Components Inhibits NF-{kappa}B Activation Induced by IL-1beta—Because NF-{kappa}B was shown to be the key mediator of colonic inflammation in amebic infection (4) and stress response is increasingly being shown to inhibit this molecule (12, 17–20), we studied the activation of NF-{kappa}B following amebic protein treatment. For this we treated M-IEC with SAP for 12 h and then stimulated with IL-1beta, a prototypical NF-{kappa}B activator. As shown, pretreatment with amebic proteins inhibited NF-{kappa}B-DNA binding activity (Fig. 6A), nuclear translocation of NF-{kappa}B p65 subunit (Fig. 6B), and I{kappa}B-{alpha} phosphorylation (Fig. 6C) induced by IL-1beta in macrophage-conditioned colonic epithelial cells.

Ameba-induced Stress Response Inhibits IKK Activity via Hsp27—Because I{kappa}B is phosphorylated by IKK, we checked IKK activity in an in vitro kinase assay. As shown in Fig. 6D, amebic pretreatment suppressed IL-1beta-induced IKK activity. Moreover, when HSF-1 gene was silenced by siRNA, this suppression was abrogated, suggesting that stress response is involved in this suppression. Thus, to precisely identify which Hsp mediates this effect, we silenced Hsp27 and 72 and checked the IKK activity. As shown in Fig. 7A, silencing Hsp27 significantly restored IKK activity, whereas Hsp72 did not have a significant effect on this suppression. We observed a significant suppression in the expression of both Hsp27 (not shown) and Hsp72 (Fig. 7B) by siRNA.


Figure 6
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  		FIGURE 6.
Amebic proteins inhibit IL-1beta-induced NF-{kappa}B activation. A, M-IEC were treated with SAP (100 µg/ml) or medium for 12 h before stimulating with 5.0 ng/ml of IL-1beta for 30 min. 10.0 µg of nuclear extract was subjected to electrophoretic mobility shift assay using NF-{kappa}B consensus oligonucleotide as described under "Experimental Procedures." The arrows indicate NF-{kappa}B-DNA complexes. B, cells were treated with IL-1beta with or without prior treatment with SAP, and nuclear extract was collected at different time points and Western blot done to check the NF-{kappa}B p65 subunit. The top panel shows p65 nuclear translocation with IL-1beta alone. The bottom panel was from cells pretreated with SAP. The membranes were stripped and reprobed with anti-histone antibody for normalization (not shown). C, Western blots of whole cell extracts showing the phosphorylation status of I{kappa}B with IL-1beta alone (top panel) or following SAP treatment and IL-1beta stimulation (bottom panel). D, M-IEC transfected or not with HSF-1 siRNA was treated with SAP or medium alone for 12 h before stimulating with IL-1beta for 30 min. In vitro kinase assay was done following immunoprecipitation with IKK-{alpha} as described under "Experimental Procedures." The top panel shows kinase assay using glutathione S-transferase-I{kappa}B as substrate. The bottom panel is Western blot of cell lysate to confirm equal quantities of IKK-{alpha}. All of the blots shown are one of two to three experiments.

 


Figure 7
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  		FIGURE 7.
Hsp27 mediates amebic protein induced inhibition of IKK activity. A, M-IEC transfected with control, Hsp27 or Hsp72 siRNA were treated with SAP for 12 h followed by 30 min of stimulation with IL-1beta. Kinase assay was done using I{kappa}B as substrate (top panel) and IKK-{alpha} level checked for normalization (bottom panel). B, M-IEC were transfected with either control or Hsp72 siRNA for 30 h as described, and the cell were extracts probed with Hsp72 antibodies.

 
Heat Shock Proteins Associate with IKK Complex in IEC and Amebic Components Enhance This Interaction—Hsp are known to associate with IKK complex and modulate its activity in other cell lines (20, 29). Hence, for the first time we checked the interaction between Hsp and IKK in intestinal epithelial cells and found that Hsp27, 60, and 90 associate with IKK-{alpha} (Fig. 8A), and treatment with SAP increased the association of Hsp27 with IKK-{alpha} by 2-fold (Fig. 8B). As expected, siRNA of Hsp27 reduced this association. Results from Figs. 7A and 8B together confirm that Hsp27 negatively regulates IKK activity. All of the Hsp tested, Hsp27, 60, 72, and 90 interacted with IKK-beta in Caco-2 IEC (Fig. 8A) and again Hsp27 interaction with IKK-beta was also enhanced by SAP treatment (data not shown).


Figure 8
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  		FIGURE 8.
Hsp associate with IKK complex in intestinal epithelial cells. A, co-immunoprecipitation of IKKs with Hsp antibodies. Macrophage-conditioned Caco-2 cell lysates were immunoprecipitated (IP) with antibodies against different Hsp ran on a reducing gel and probed with anti-IKK-{alpha} or IKK-beta antibody. Whole cell lysates checked for IKK-{alpha} and IKK-beta expression (bottom two panels). B, amebic proteins enhance the interaction between Hsp27 and IKK-{alpha}. M-IEC transfected or not with Hsp27 siRNA were treated with SAP (100 µg/ml) for 12 h, lysates co-immunoprecipitated with Hsp27 and checked for IKK-{alpha} by SDS-PAGE and Western blot. The relative densitometric values of the bands are shown. Equal quantity of cell lysates was probed with Hsp27 and IKK-{alpha} (bottom panels). All of the experiments were repeated two to three times and representative Western blots for each are shown. IP, immunoprecipitation; IB, immunoblot.

 


Figure 9
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  		FIGURE 9.
NF-{kappa}B inhibition by SAP requires phosphorylation of Hsp27. A, conditioned IEC were transfected with vector (Vec), wild type (WT), or mutant Hsp25 plasmids during co-culture as described, and whole cell lysates were subjected to SDS-PAGE and probed with antibodies against hamster Hsp25 and actin. B, M-IEC overexpressing hamster Hsp25 plasmids were treated with SAP for 12 h, and lysates were subjected to immunoprecipitation using anti-hamster Hsp25 or control rabbit IgG antibody followed by Western blot using IKK-{alpha} antibody. Cell lysates from the same cells were probed with IKK-{alpha}. C, conditioned IEC transfected with vector, wild type, or mutant Hsp25 plasmids were pretreated or not with SAP and IL-1beta, and whole cell lysates were checked for phospho-I{kappa}B-{alpha} by Western blot as described. Bottom panel, 10 µg of nuclear extracts from cells treated similarly were probed with anti-p65 antibody. D, M-IEC treated with SAP for different time periods and cell lysates probed with Ser(P)15-Hsp27 and actin. Representative blots from at least two different experiments were shown. IB, immunoblot.

 


Figure 10
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  		FIGURE 10.
Amebic proteins confer protection against oxidative and apoptotic injuries. A, M-IEC were treated with SAP (100 µg/ml) for 12 h and then incubated with 10 mM of hydrogen peroxide for further 12 h, and cell viability was checked by neutral red assay as described. **, p < 0.01 compared with control; *, p < 0.05 compared with H2O2 alone. NS, not significant; Ctl, control. B, M-IEC (T84 cells) were exposed to SAP (100 µg/ml) for 12 h before treating with 1.0 µg/ml of Fas L for 12 h. Cleavage of Pro caspase-3 was examined by Western blot. The experiment was repeated three times, and one representative blot is shown.

 
Phosphorylation of Hsp27 Is Required for Interaction with and Inhibition of IKK—To confirm that Hsp27 mediated IKK inhibition in IEC and to fully understand the mechanism involved, we overexpressed wild type and phosphorylation-deficient mutant of hamster Hsp25 (AA) in conditioned epithelial cells (Fig. 9A). Transfected M-IEC was treated with SAP and checked for interaction between Hsp25 and IKK-{alpha}. As shown in Fig. 9B, although overexpression of wild type Hsp25 enhanced SAP-induced association with IKK-{alpha}, Hsp25AA significantly inhibited this interaction. Consistent with the negative regulation of IKK by Hsp27, this decreased interaction between IKK-{alpha} and Hsp27 abrogated the inhibitory effect of SAP on IKK activity, seen as restored I{kappa}B phosphorylation and NF-{kappa}B p65 nuclear translocation (Fig. 9C). Consistently, we observed that SAP induced a delayed but significant phosphorylation of Ser15 Hsp27 in M-IEC (Fig. 9D).

Ameba-induced Stress Response in Conditioned Epithelial Cells Has Cytoprotective Function—Because Hsp also have cytoprotective abilities in different cells, we checked whether amebic proteins can protect IEC against injuries caused by diverse agents. As shown in Fig. 10A, pretreatment with SAP significantly increased cell viability following oxidative injury by hydrogen peroxide and reduced caspase-3 cleavage induced by the apoptotic agent, Fas L (Fig. 10B).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we show that SAP have protective effects mediated by Hsp on intestinal epithelial cells. Interestingly, the Hsp were induced only in epithelial cells that have been exposed to macrophage secretions for 24 h but not in the naive IEC. We made two novel observations: first, this is the first report on the protective effect of amebic proteins, and second, macrophage secretions can prime epithelial cells to elicit stress responses.

Co-culture systems have been popular because of their better simulation of in vivo situation wherein epithelial and immune cells exist in close proximity and respond to each other components. Several studies reported a differential epithelial response, mostly with respect to cytokines, following exposure to immune cells. Two recent studies reported the induction of Hsp25 and Hsp72 in mouse intestinal epithelial cells co-cultured with lymphocytes, and IL-2 was found to be the mediator (13, 16). We made the novel observation that intestinal epithelial cells express Hsp in response to amebic proteins following exposure to macrophage secretions.

Notably, basal Hsp expression remains unchanged in macrophage-conditioned IEC, suggesting that epithelial cells are only primed rather than activated by macrophage secretions. The cross-talk between macrophages and IEC could be bi-directional in the sense that macrophages also respond to epithelial secretions such as monocyte chemotactic protein-1 and transforming growth factor-beta (30). However, amebic proteins could induce Hsp72 in epithelial cells that have been incubated with macrophage secretions without co-culturing, suggesting that epithelial cell modification of macrophages is not required to elicit a differential epithelial response. Because differentiated THP-1 cells are shown to constitutively produce a variety of cytokines such as TNF-{alpha}, IL-1beta, IL-6, IL-8, etc. (31), and IL-1beta induce Hsp expression (32), we checked the role of these cytokines in priming the IEC. Adding neutralizing antibodies against TNF-{alpha} and IL-1beta in the macrophage medium did not significantly inhibited ameba-induced Hsp expression (data not shown). Previous studies (33) have shown that co-culture with activated peripheral blood mononuclear cells altered T84 epithelial cell physiology and that exogenously added TNF-{alpha} or interferon-{gamma} did not mimic the changes induced by immune cells. This suggests that mediators in immune cell secretions have an effect (both adverse and beneficial) on epithelial responses, but their identities are not known.

IEC are known to express Hsp in response to diverse insults such as pathogens, pathogen-specific molecules, and chemicals, and immune cells can alter this response. Accordingly, we checked the expression of four Hsp belonging to different families and found specific up-regulation of Hsp27 and 72 but not Hsp60 and 90 (data not shown) by SAP. Our observation of involvement of ERK MAP kinase in ameba-induced Hsp expression is consistent with previous reports (16, 34, 35) and extends the current thinking that MAP kinases play an important role in regulating both inflammatory and stress pathways. After establishing that amebic proteins induce Hsp in macrophage primed epithelial cells by activating HSF-1, we proceeded to check the functional significance of this phenomenon. Because our objective was to understand how colonic inflammation was absent in the majority of infected individuals and given the evidence that Hsp suppress NF-{kappa}B activation, we critically analyzed whether and how Hsp induced by amebic proteins inhibits IL-1beta-mediated NF-{kappa}B activation. We chose IL-1beta for two reasons: first, is it is a prototypical NF-{kappa}B inducer, and second, it was shown to play a role in ameba-induced colitis (2, 36). Moreover, inhibiting NF-{kappa}B significantly reduces amebic colitis in a mouse model of intestinal amebiasis (4). NF-{kappa}Bis a ubiquitous transcription factor that regulates a number of genes involved in inflammation and immune response (37). Activation of this transcription factor is critically regulated at multiple steps. Recently, the inhibitory effects of Hsp on NF-{kappa}B activation are increasingly being demonstrated in different cell systems. Hsp72 has been found to associate with the p65 subunit of NF-{kappa}B and inhibits the nuclear transport of the latter in T-cells (38), and Hsp27 has been shown to be a ubiquitin-binding protein regulating the degradation of I{kappa}B expression, thereby indirectly influencing NF-{kappa}B activation (39). Recent studies show that the IKK complex is the potential target for Hsp inhibition of NF-{kappa}B pathway (17, 18, 40). In particular, one study showed that Hsp27 interacts with IKK complex and negatively regulates its activation by TNF-{alpha} (20) in HeLa cells. Because SAP increased Hsp72 expression and inhibited IL-1beta-induced NF-{kappa}B p65 nuclear translocation, we checked for their interaction in IEC but failed to see any association (data not shown). We found that I{kappa}B phosphorylation was also inhibited, which was a direct result of reduced IKK activity by SAP treatment. siRNA technology has rapidly become a revolutionary tool for efficient silencing of gene expression in a variety of experimental settings (41). We exploited this powerful system to silence Hsp genes to understand their role in ameba mediated suppression of IKK activation and found that silencing HSF-1 or Hsp27 but not 72 resulted in significant abrogation of this inhibition. Because different Hsp interact with and regulate IKK complex, we checked whether amebae-induced Hsp interacted with the IKK subunits in intestinal epithelial cells. For the first time, we reported an association between IKK and Hsp in IEC. Hsp27, 60, and 90 constitutively interact with IKK-{alpha}, and the interaction of Hsp27 with IKK-{alpha} was strongly enhanced following SAP treatment. Although Hsp27 and Hsp90 have been previously shown to associate with the IKK complex, this is the first report of the interaction between Hsp60 and IKK. Our observation that all Hsp tested interacted with IKK-beta is surprising but could be attributed to the use of mild detergent (CHAPS) in the cell lysis buffer for co-immunoprecipitation. We found that Hsp27 association with IKK-beta was also increased by amebic protein treatment (not shown). A previous report (20) showed that Hsp27 association with IKK-beta but not with IKK-{alpha} increased in response to TNF-{alpha}. The reason(s) for this selective interaction is unclear.

Because post-translational modifications of Hsp27 are known to regulate its biological activity (20), we tested the role of phosphorylation of Hsp27 in its ability to inhibit NF-{kappa}B activation. Overexpression of the phosphorylation-deficient mutant of hamster Hsp25 (at serines 15 and 90) in IEC resulted in significant reversal of NF-{kappa}B inhibition by amebic proteins. There are two sites phosphorylated in hamster Hsp27 (Ser15 and Ser90) and three in human (Ser15, Ser78, and Ser82). Ser78–Ser82 of human Hsp27 probably works in tandem as the equivalent of the unique Ser90 in hamster Hsp25. We also observed that SAP induces significant phosphorylation of Ser15 (Fig. 9D) but not Ser72 (data not shown) after prolonged treatment in M-IEC. Because Hsp27 phosphorylation depends on p38 MAP kinase (20), this could have resulted from a delayed activation of p38 MAP kinase, as we did not find either p38 (data not shown) or Hsp27 phosphorylation in the early time periods of 1 h. Nonetheless it is clear that overexpression of Hsp27 amplified the SAP effects, whereas blocking the Hsp27 phosphorylation significantly abrogated the amebic inhibitory effect on NF-{kappa}B activation. These studies confirm and further strengthen the concept that post-translational modifications, particularly, the phosphorylation of Hsp27 plays an important role in NF-{kappa}B regulation via inhibiting IKK activity. Extending our observations on the protective effects of ameba-induced stress responses, we also showed that prior treatment with amebic proteins protected the epithelial cells against hydrogen peroxide and Fas L treatment. This is not surprising in view of the potent apoptotic inhibitory effects of Hsp72 (42, 43). In fact, both Hsp27 and 72 are powerful chaperones and inhibit key effectors of the apoptotic machinery including apoptosome, caspase activation complex, and apoptosis-inducing factor (44). Consistently, we also observed increased cell survival following apoptotic stimuli in M-IEC pretreated with amebic proteins. Although this observation supports the anticipated protective effects of stress response, its functional significance with respect to ameba pathogenesis is difficult to surmise. Contact-dependent apoptosis induction by E. histolytica was reported in immune cells such as neutrophils, T-cells, and erythrocytes (23, 45, 46). It was also shown that E. histolytica preferentially ingests apoptotic Jurkat T-cells. But hitherto no data are present on epithelial cell apoptosis by the parasite. Assuming a similar phenomenon for epithelial cells, it is reasonable to argue that stress response by the host is an efficient way to circumvent parasite-induced cell death. At present, it is not known what soluble amebic molecule(s) is responsible for the induction of Hsp. In summary, we have shown that amebic proteins can inhibit NF-{kappa}B activation and promote cell survival via stress protein expression in macrophage primed intestinal epithelial cells (Fig. 11). These studies for the first time, demonstrate a potential mechanism by which intestinal inflammation induced by E. histolytica might be inhibited in majority of infected individuals and suggests that amebic colitis could result from the lack of such protective responses to suppress pro-inflammatory cytokine induction in a minority of susceptible individuals.


Figure 11
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  		FIGURE 11.
Schematic model for suppression of amebic colitis. Soluble amebic proteins activate the heat shock transcription factor-1 to form trimers from its inactive monomers. Activated HSF-1 induces the expression of Hsp27 and Hsp72, which perform different functions. Phospho-Hsp27 associates with IKK and inhibits its activity, thereby suppressing NF-{kappa}B activation induced by the proinflammatory cytokines released in response to amebic infection. Suppression of NF-{kappa}B favors increased apoptosis. However, Hsp72, which apparently does not play a role in NF-{kappa}B signaling in IEC, might function to promote cell survival through its potent anti-apoptotic activity. Together the stress response induced by amebic proteins functions to inhibit intestinal inflammation and promote cell survival.

 

   FOOTNOTES
 
* This work was supported by grants from the Canadian Institutes of Health Research and the Natural Sciences and Engineering Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Recipient of a McGill University Graduate Fellowship. Back

2 To whom correspondence should be addressed: University of Calgary, Faculty of Medicine, Dept. of Microbiology & Infectious Diseases, 3330 Hospital Dr. NW, Calgary, AB T2N 4N1, Canada. Tel.: 403-210-3975; Fax: 403-270-2772; E-mail: kchadee{at}ucalgary.ca.

3 The abbreviations used are: IL, interleukin; SAP, soluble amebic protein(s); Hsp, heat shock protein(s); siRNA, small interfering RNA; IKK, I{kappa}B kinase; IEC, intestinal epithelial cell(s); MAP, mitogen-activated protein; MAPK, MAP kinase(s); CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; ERK, extracellular signal-regulated kinase; HSF, heat shock factor; M-IEC, macrophage-conditioned IEC; TNF, tumor necrosis factor. Back


   ACKNOWLEDGMENTS
 
We thank Elaine De Heuvel for excellent technical assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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