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Istituto Nazionale di Ricerca per gli Alimenti e la Nutrizione (INRAN), Via Ardeatina 546, 00178 Roma, Italy
3To whom correspondence should be addressed. E-mail: mengheri{at}inran.it.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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and tumor necrosis factor-, and reduced that of the anti-inflammatory cytokine transforming growth factor-ß, compared with uninfected cells. The addition of 0.2 or 1 mmol/L ZnO counteracted the alteration of cytokine mRNA levels caused by ETEC. The protective effects of ZnO were not due to any antibacterial activity, because the viability of ETEC grown in a medium containing ZnO was unaffected. In conclusion, ZnO may protect intestinal cells from ETEC infection by inhibiting the adhesion and internalization of bacteria, preventing the increase of tight junction permeability and modulating cytokine gene expression.
KEY WORDS: • Zinc oxide • Caco-2 cells • enterotoxigenic Escherichia coli • protective effect • intestinal cells
The role of zinc in the anti-inflammatory response and resistance to infection is well known (1,2). In particular, zinc oxide (ZnO) appears to have a strong protective effect in resisting intestinal diseases. Several studies conducted on piglets show that dietary ZnO supplementation may prevent or alleviate diarrhea, which is mainly caused by enterotoxigenic Escherichia coli (ETEC),3 strain K88 (3–5). Studies also report that pigs fed high dosages of ZnO maintain stable levels of the intestinal flora and diverse coliforms that may compete with diarrhoegenic strains for colonization sites (6). Moreover, reduced translocation of pathogenic bacteria from the small intestine to the mesenteric lymph nodes has been reported in pigs fed supplemental ZnO (4). The amounts of ZnO used in these studies greatly exceeded physiological requirements. However, there is evidence that pharmacological levels of ZnO improve piglet growth performance and are less toxic than other inorganic zinc supplements (7,8). In addition, studies evaluating the healing of skin wounds in pigs found that ZnO is more effective than zinc sulfate (ZnSO4) in promoting healing; topical ZnO enhances reepithelialization, whereas ZnSO4 is ineffective (9).
It has been suggested that the successful prophylactic use of ZnO in preventing diarrhea may be due to a bactericidal effect of zinc. In fact, studies conducted in vitro report that ZnO inhibits bacterial growth (10,11). However, studies conducted with pigs fed supplemental ZnO also report that intestinal E. coli levels are unaffected (3,6). Despite these studies, very little is known about the mechanisms of the ZnO protective effect.
The integrity of the intestinal barrier is fundamental to the proper functioning of the epithelial cells and to preventing the entry of pathogenic bacteria that cause inflammation (12). Some authors report that zinc plays a role in maintaining epithelial barrier integrity and function. In fact, they show that zinc deficiency alters the barrier function of porcine endothelial cells, whereas treatment of these cells with zinc prevents TNF-induced disruption of the cell monolayer (13,14). In addition, zinc supplementation may improve mucosal repair and paracellular permeability in experimental colitis (15). Whether ZnO is able to protect membrane integrity from potential damage by E. coli remains to be elucidated.
The present study attempted to better investigate the potential benefits of ZnO in reducing ETEC infection of intestinal cells and the related mechanisms, by using a well-characterized model of human enterocytes, namely Caco-2 cells. These cells can undergo spontaneous differentiation and achieve the characteristics of mature enterocytes, such as structural polarization, tight junctions and apical microvilli (16). Caco-2 cells have been widely used to study interactions of different bacteria with host epithelial cells (17–20).
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The human intestinal Caco-2 cell line (obtained from Monique Rousset, Institut Biomedical de Cordeliers, Paris, France) was used between passages 80 and 100. Cells were routinely grown in plastic tissue culture flasks (75 cm2 growth area, Becton Dickinson, Milan, Italy) in DMEM (3.7 g NaHCO3/L, 4 mmol glutamine/L, 100 g heat-inactivated fetal calf serum/L, 10 g nonessential amino acids/L, 105 U penicillin/L and 100 mg streptomycin/L). All cell culture reagents were from Biochrom (Milan, Italy). The cells were maintained at 37°C in an atmosphere of 5% CO2/95% air at 90% relative humidity. For experiments with ZnO and ETEC, cells were seeded on tissue culture plates or 12-mm, 0.4-µm pore Transwell filters (polyethylene terephtalate filter inserts for cell culture; Becton Dickinson), as described below. After confluency, cells were left for 15 to 17 d to allow differentiation, as previously reported (21). The medium was changed 3x/wk.
Bacterial growth.
The ETEC strain K88 (provided by Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia, Reggio Emilia, Italy) was grown in Luria broth (LB) containing 10 g tryptone/L, 5 g yeast extract/L and 10 g NaCl/L, pH 7.0. Tryptone and yeast extract were from OXOID (Basingstoke, UK). After overnight incubation at 37°C with shaking, bacteria were diluted 1:100 in fresh LB and grown to midlog phase. Bacterial concentration was determined by densitometry and confirmed by serial dilution followed by viable plate counts on LB agar after overnight incubation at 37°C. For experiments with Caco-2 cells, bacteria were harvested by centrifugation at 3000 x g for 10 min at 4°C, then resuspended in antibiotic-free DMEM. The viability of ETEC grown on DMEM did not differ from that of bacteria grown on LB medium, as tested in preliminary experiments.
Bacterial survival in medium containing ZnO.
Because it has been reported that ZnO can exert an antimicrobial effect, ETEC was grown in LB medium containing a high concentration of ZnO (1 mmol/L; Sigma, Milan, Italy). The number of viable bacteria was determined by agar plating.
Tight junction integrity.
Tight junction permeability was determined with Caco-2 cells differentiated on Transwell filters (5 x 105 cells/filter) by transepithelial electrical resistance (TEER) and paracellular flux of the radiolabelled marker 14C-inulin, as previously reported (21). The potential toxicity of ZnO was tested by adding several ZnO concentrations (0.01 to 5 mmol/L) to the apical compartment of the Transwell filter for several hours (0 to 31 h) or days (0 to 14 d). The TEER was monitored with a Millicell Electrical Resistance System (Millipore, Bedford, MA) and expressed as 
·cm2; the filter resistance was subtracted from the reading, and the result was multiplied by the surface area of the monolayer. The TEER was checked before each experiment, and only cell monolayers with TEER > 1000 ·cm2 were used.
Transfer of 14C-inulin was assayed after treatment with ZnO (1 or 5 mmol/L) for 24 h, by adding 3.7 MBq of 14C-inulin (444 MBq/mmol; Perkin Elmer, Mechelen, Belgium) to the apical compartment for the last 2 h of incubation. Aliquots of basolateral medium were collected, and radioactivity was assessed with a scintillation counter (Beckman, Milan, Italy). Passage of 14C-inulin was expressed as a percentage of the total radioactivity applied.
The effect of ETEC on membrane integrity was tested by infecting cells with 0.5 mL of DMEM containing 5 x 107 bacteria, to achieve a multiplicity of infection of 100 (bacteria/Caco-2 cell ratio). The protective effect of ZnO was tested by adding various concentrations of ZnO (0.05, 0.1, 0.2, 0.5 or 1 mmol/L) with ETEC infection. Membrane integrity was assayed by measuring TEER every 30 min from 0 to 4.5 h of treatment and by measuring 14C-inulin transfer after 1.5, 3 and 4.5 h of treatment. These assays were not continued further, because membrane permeability was markedly damaged after 4.5 h of ETEC infection.
The ZnO dilutions were prepared in DMEM from a stock solution of 400 mmol ZnO/L dissolved in 5% acetic acid. Preliminary experiments indicated that the concentration of acetic acid in the ZnO dilutions did not affect tight junction integrity. Because previous studies reported that the bioavailability of zinc from ZnO is lower than that of zinc from other compounds, such as ZnSO4 (22,23), we also performed preliminary experiments to verify the effective uptake of zinc. Zinc accumulation both in the basolateral medium and inside the cells was measured after treatment with 1 and 5 mmol/L concentrations of ZnO and ZnSO4, using flame atomic absorption spectrophotometry as previously reported (21). Zinc accumulation in the basal compartment increased with increasing concentration of ZnO, and accumulation inside the cells increased only with 5 mmol ZnO/L, indicating effective zinc uptake (data not shown). The uptake of ZnSO4 did not differ from that of ZnO, in agreement with recent studies (24).
Bacterial adhesion.
Caco-2 cells were differentiated in 24-well plates (1 x 106 cells/well), then infected with 1 mL of DMEM containing 1 x 108 bacteria. Inhibition of adherence was tested by adding various concentrations of ZnO (0.05, 0.2 or 1 mmol/L) to the infected cells. After incubation at 37°C for 1.5 h, nonadherent bacteria were removed by five washes with HBSS (137 mmol/L NaCl, 5.36 mmol/L KCl, 1.67 mmol/L CaCl2, 1 mmol/L MgCl2, 1.03 mmol/L MgSO4, 0.44 mmol/L KH2PO4, 0.34 mmol/L Na2HPO4 and 5.6 mmol/L glucose). Cells were then lysed with 1% Triton-X-100, and viable bacteria were quantified by plating appropriate serial dilutions of lysates on LB agar.
Bacterial internalization.
Invasion of epithelial cells by viable bacteria was evaluated by gentamicin protection assay, as previously described (17). Caco-2 cells were differentiated in 24-well plates (1 x 106 cells/well), then infected with 1 mL of DMEM containing 1 x 108 bacteria and incubated for 1.5 h. The inhibition effect of ZnO was tested by treating cells with both ETEC and various concentrations of ZnO (0.05, 0.2 or 1 mmol/L) for 1.5 h, then incubating with culture medium containing gentamicin sulfate (50 mg/L; Sigma), for 2.5 h to kill residual viable extracellular bacteria. Viable intracellular bacteria were quantified by agar plating.
Cytokine mRNA.
Caco-2 cells were differentiated in 6-well plates (4 x 106 cells/well, then treated with ETEC (4 x 108 bacteria/well) and ZnO (0.05, 0.2 or 1 mmol/L), either separately or together in 4 mL of DMEM, for 2 h. This time was chosen because preliminary experiments indicated that alterations in cytokine mRNA levels were most evident after 2 h of treatment. Total RNA was extracted with TRIZOL reagent (Life Technologies, GIBCO BRL, Milan, Italy) and 1 µg of RNA was used for RT-PCR treatment to analyze for mRNA-encoding IL-8, growth-related oncogene (GRO)-, TNF- and transforming growth factor (TGF)-ß, as previously reported (25). Each cytokine was coamplified with glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) as an internal control. The sequences of primers were as follows: IL-8 sense 5'-ATT TCT GCA GCT CTG TGT GAA-3' and antisense 5'-TGA ATT CTC AGC CCT CTT CAA-3' (expected fragment size of 289 bp); GRO- sense 5'-TGA GCC CCA TGG CCC GCG CTG-3' and antisense 5'-CCC TTC TGG TCA GTT GGA TTT GTC AC-3' (expected fragment size of 341 bp); TNF- sense 5'-CAG AGG GAA GAG TTC CCC AG-3' and antisense 5'-CCT TGG TCT GGT AGG AGA CG-3' (expected fragment size of 324 bp); TGF-ß sense 5'-CTC CGA GAA GCG GTA CCT GAA-3' and antisense 5'-CAC TTG CAG TGT GTT ATC CCT-3' (expected fragment size of 288 bp); GAPDH sense 5'-TGA AGG TCG GAG TCA ACG GAT TTG GT-3' and antisense 5'-CAT GTG GGC CAT GAG GTC CAC CAC-3' (expected fragment size of 977 bp). The primers were provided by MWG Biotech (Florence, Italy). The PCR products were analyzed on agarose gel, and the relative intensity of the bands was measured with Scion imaging software (Scion, Frederick, MD). The relative mRNA levels were evaluated using the ratio of cytokine/GAPDH mRNA intensity.
Statistical analysis.
The significance of the differences was evaluated by one-way ANOVA followed by Fisher’s test at fixed time points. The effects of ZnO concentration and treatment time on TEER were evaluated by two-way ANOVA followed by Tukey’s test (Fig. 1A). The homogeneity of the slopes of linear regressions and the effects of time, treatments and time-treatment interactions on TEER were evaluated by the general linear model followed by Tukey’s test (Fig. 2A). Variances were homogeneous. Differences were considered significant with P < 0.05. Linear regression analysis was used to determine the correlation coefficient (r) used to evaluate the linear relationship between ZnO concentration and the number of adhesive (Fig. 3) and internalized bacteria (Fig. 4). All statistical analyses were performed with SAS release 8.1 statistical software (SAS Institute, Cary, NC)
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Concentrations of 0.01 and 1 mmol/L ZnO did not affect TEER, even after 31 h of treatment, and 5 mmol/L ZnO increased TEER slightly, compared with that of untreated control cells (Fig. 1A, P < 0.01). In long-term ZnO treatment (up to 14 d), only cells treated with 5 mmol/L ZnO had lower TEER than that of untreated control cells from d 5 of treatment onward (data not shown). Treatment with 1 mmol/L ZnO did not affect 14C-inulin transfer, compared with control cells, whereas treatment with 5 mmol/L ZnO increased inulin transfer (Fig. 1B). However, the rate of 14C-inulin passage was still very low (0.085%), and this result does not indicate an increase in tight junction permeability, as confirmed by the increase in TEER with this treatment. Indeed, only marked increases in inulin passage have been reported in association with increased tight junction permeability (26).
Membrane damage induced by ETEC and protective effect of ZnO.
The ETEC induced increased tight junction permeability, because TEER decreased with time after infection (Fig. 2A; P < 0.001) and 14C-inulin transfer was slightly greater at 1.5 h after infection (P < 0.05), and much greater at 3 and 4.5 h after infection (both P < 0.001), compared with untreated control cells (Fig. 2B). The ZnO treatment inhibited the ETEC-induced membrane damage, and this effect was time and dose dependent. Indeed, the TEER of cells treated with both ETEC and 0.05 or 0.1 mmol/L ZnO was lower than that of control cells (Fig. 2A, P < 0.001). The TEER of cells treated with both ETEC and 0.2, 0.5 or 1 mmol/L ZnO did not differ from that of control cells. The 14C-inulin transfer assay confirmed the protective effect of ZnO (Fig. 2B). Indeed, the increased 14C-inulin passage caused by ETEC was completely inhibited by 0.2 and 1 mmol/L ZnO (P < 0.001), whereas 0.05 mmol/L ZnO partially inhibited the great increase in inulin passage at 4.5 h after infection (P < 0.05).
Reduction in ETEC adhesion.
A high percentage of ETEC adhered to Caco-2 cells at 1.5 h after infection (Fig. 3). Treatment with 0.2 or 1 mmol/L ZnO reduced bacterial adhesion, compared with infected cells, but 0.05 mmol/L ZnO was ineffective. The correlation coefficient (r = 0.91) indicated a positive (P < 0.01) dose-response correlation between ZnO concentration and reduction in number of adhered bacteria.
Anti-invasive effect of ZnO on ETEC.
The ETEC invaded Caco-2 cells after 2.5 h of challenge, although only small numbers of the total bacteria were internalized (Fig. 4). Treatment with ZnO induced a dose-dependent anti-invasive effect. Indeed, compared with infected cells, treatment with 0.2 and 1 mmol/L ZnO markedly reduced and almost totally inhibited intracellular bacterial invasion, respectively, whereas 0.05 mmol/L ZnO was ineffective. Gentamicin treatment efficiently killed the bacteria. The correlation coefficient (r = 0.94) indicated a positive (P < 0.05) dose-response correlation between ZnO concentration and number of internalized bacteria.
Cytokine gene expression.
Expression of IL-8, GRO-, TNF- and TGF-ß in cells treated with various concentrations of ZnO did not differ from that of untreated control cells (data not shown). Exposure to ETEC infection for 2 h induced upregulation of the inflammatory cytokines IL-8, GRO- and TNF- and downregulation of anti-inflammatory cytokine TGF-ß, compared with control cells (Fig. 5; P < 0.001). Treatment with 0.2 or 1 mmol/L ZnO together with ETEC infection prevented the alterations in cytokine expression induced by ETEC. In cells treated with 0.05 mmol/L ZnO together with ETEC infection, the expression of IL-8 and TNF- was lower than that of infected cells (P < 0.01), but higher than that of control cells, whereas the expression of TGF-ß and GRO- did not differ from that of infected cells.
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The number of colony-forming units of ETEC grown in medium containing ZnO (1 mmol/L) did not differ from that of ETEC grown without ZnO after incubation for 2 and 4 h (Fig. 6), indicating that ZnO did not affect the viability of ETEC (P < 0.001).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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There is an opinion that the protective effect of ZnO is due to its antibacterial activity. This is based on observations that ZnO inhibits the growth of bacterial species commonly involved in endodontic infections, such as Peptostreptococcus micros, Streptococcus intermedius, Enterococcus faecalis, and Porphyromonas gingivalis (27). In addition, recent in vitro studies show that ZnO inhibits the growth of Staphylococcus aureus and E. coli (11). However, studies with pigs fed supplemental ZnO report no effect on the number of excreted E. coli (3,6). Our results with ETEC grown in a medium containing ZnO show that ZnO did not kill the bacteria, indicating that the protective effect of ZnO is not ascribable to an antibacterial effect per se. This finding is in agreement with the fact that gram-positive bacteria are more susceptible to ZnO than gram-negative bacteria (10). Thus, we conclude that ZnO may have a bactericidal effect on some bacterial species but not on E. coli K88.
The strain of ETEC used in this study adheres to the brush border of isolated human duodenal enterocytes (28) as well as to Caco-2 cells (29). Attachment to mucosal surfaces is the first step in the pathogenesis of this bacterium, which is related to the release of enterotoxins (30). Thus, the inhibitory effect of ZnO on bacterial adhesion is of great importance in preventing ETEC infection. The K88 fimbrial adhesins are filamentous surface appendages with which the bacterium attaches to mucosal specific receptors (30). A recent study shows that the type 1 fimbriae of E. coli K12 bind to zinc, and specifically to only the oxide form of zinc (31). Supposing a similar capacity for K88 fimbriae, we suggest that ZnO reduces ETEC adhesion by binding to the K88 fimbriae, thus interfering with the recognition of K88 by the specific receptors on intestinal cells.
Zinc plays a fundamental role in maintaining membrane function and stability. Some authors suggest that the mechanism of this activity involves the stabilization of the membrane structure (32) or the displacement of redox-active metals to prevent free-radical oxidative damage (33,34). Hennig et al. showed that zinc is vital to cell integrity and barrier function, using porcine endothelial cells cultured in zinc-deficient or zinc-supplemented media (13). These authors also found that treatment with zinc protects the cells against TNF-–induced disruption of the cell monolayer (14). Other authors report that feeding supplemental zinc to rats with experimental colitis improves mucosal repair by regulating tight junction permeability (15). In agreement with these data, our findings show that ZnO may prevent the increase in tight junction permeability induced by ETEC, as indicated by both the high TEER values and the absence of 14C-inulin transfer.
The consequences of increasing tight junction permeability can be dramatic, as it can allow the indiscriminate entry of extracellular antigens as well as of pathogenic bacteria. It was recently suggested that the lateral enterocyte surface may be the preferential site of entry for some strains of bacteria (35,36). Wells et al. reported that treatment of Caco-2 cells with cytochalasin to induce actin disruption augmented internalization of E. coli, Salmonella typhimurium and Proteus mirabilis by a mechanism that appeared to involve exposure of the enterocyte lateral surface (35). Other authors found that the lateral surface of mature Caco-2 cells appeared to be the preferred entry site of Listeria monocytogenes following disruption of the tight junctions by a calcium-free medium (36). Although not considered an invasive pathogen, ETEC is reported to enter cultured human epithelial cells (37). Our results show that a certain percentage of ETEC K88 was able to invade Caco-2 cells after 2 h of exposure, when the tight junctions were damaged. Thus, the ability of ZnO to prevent the disruption of tight-junction integrity may contribute to the inhibition of bacterial internalization through this entry site. Whether this is achieved through prevention of actin depolymerization by ZnO remains to be determined.
Recently, many cytokines have been shown to regulate tight-junction functioning in epithelial and endothelial cells, although the mechanisms are poorly understood (38,39). The cytokine TNF- can act synergistically with INF-
to induce structural changes in tight junctions, whereas TGF-ß upregulates the barrier function and counteracts the IFN- effect. Besides these activities, it is well known that cytokines play a fundamental role in the inflammatory response. The common response to infection is infiltration of the affected tissue by inflammatory cells. The cytokine IL-8 is considered one of the principal mediators in inflammation and, together with the related chemotactic GRO-, is a strong inducer of neutrophils (40). Conversely, TGF-ß is a potent anti-inflammatory cytokine (41). In the present study, the expression of IL-8, GRO- and TNF- was markedly higher, and that of TGF-ß lower, in ETEC-infected cells than in uninfected cells. Although ZnO did not completely inhibit bacterial adhesion, it counteracted the alteration of cytokine expression caused by ETEC, because the mRNA levels of IL-8, GRO-, TNF- and TGF-ß after ZnO treatment (0.2 and 1 mmol/L) did not differ from those of control cells. The fact that ZnO did not change the cytokine mRNA levels of uninfected cells suggests that ZnO affects the synthesis of these mRNAs indirectly, likely by interfering with the stimulus of the adhered bacteria to induce cytokine synthesis. Thus, in agreement with the recognized role of zinc in modulating cytokine expression in infectious disease (1), ZnO might prevent the cytokine-induced inflammatory response in infected cells and likely the consequent disruption of membrane integrity.
Several studies report that dietary ZnO supplementation improves the growth rate of young pigs and reduces the incidence of diarrhea, which is known to be caused mainly by ETEC K88 (5–8). These studies initiated ZnO supplementation at weaning, that is, when pigs are particularly exposed to bacterial infection. Thus, our model of exposing cells to ETEC together with ZnO mimics the in vivo conditions of ZnO treatment for infectious disease prevention. The mechanism of the ZnO protective effect is largely unknown. Our finding that ZnO inhibits ETEC K88 invasiveness contributes to understanding the mechanisms through which ZnO prevents ETEC-induced damage in vivo. ZnO is preferable to other inorganic forms of zinc because of its lower toxicity and higher efficiency in preventing infection (3,4). In our experiments, the concentrations of ZnO needed to prevent ETEC-induced cell damage greatly exceeded physiological requirements, but proved to be nontoxic to the cells. It is important to note that pharmacological concentrations of zinc were also necessary to induce a protective effect in vivo. Although it is difficult to compare in vitro and in vivo dosages, our results suggest that ZnO is a valid nontoxic agent for the control of ETEC infection.
In conclusion, ZnO protects cells from ETEC-induced damage by inhibiting bacterial adhesion and internalization, preventing the disruption of barrier integrity and modulating cytokine expression, not by a direct antibacterial effect.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 These authors contributed equally to this work.
4 Abbreviations used: ETEC, enterotoxigenic Escherichia coli; GAPDH, glyceraldehyde-3-phosphate-dehydrogenase; GRO, growth-related oncogene; LB, Luria broth; TEER, transepithelial electrical resistance; TGF, transforming growth factor.
Manuscript received 31 July 2003. Initial review completed 21 August 2003. Revision accepted 17 September 2003.
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6 separate experiments. Within each cytokine, means without a common letter differ (one-way ANOVA, P < 0.01).