Probiotics and Commensals Reverse TNF-α– and IFN-γ–Induced Dysfunction in Human Intestinal Epithelial Cells
2006; Elsevier BV; Volume: 130; Issue: 3 Linguagem: Inglês
10.1053/j.gastro.2005.12.015
ISSN1528-0012
AutoresSilvia Resta‐Lenert, Kim E. Barrett,
Tópico(s)Helicobacter pylori-related gastroenterology studies
ResumoBackground & Aims: Commensal bacteria are crucial for the development of the mucosal immune system. Probiotics are commensals with special characteristics and may protect mucosal surfaces against pathogens. Pathogens cause significant phenotypic alterations in infected epithelial cells, and probiotics reverse these deleterious responses. We hypothesized that probiotics and/or commensals may also reverse epithelial damage produced by cytokines. Methods: Human intestinal epithelial cells were exposed basolaterally to interferon (IFN)-γ (103 U/mL) or tumor necrosis factor (TNF)-α (10 ng/mL) for up to 48 hours and assessed for ion transport, transepithelial resistance (TER), and epithelial permeability in the presence or absence of probiotics (Streptococcus thermophilus [ST] and Lactobacillus acidophilus [LA]), or the commensal, Bacteroides thetaiotaomicron (BT). Results: Agonist-stimulated chloride secretion was inhibited by IFN-γ, an effect prevented by ST/LA or BT. The ability of ST/LA or BT to restore Cl− secretion was blocked by inhibitors of p38 MAPK, ERK1, 2, and PI3K. The cystic fibrosis transmembrane conductance regulator (CFTR) and the NKCC1 cotransporter were down-regulated by IFN-γ, and ST/LA pretreatment reversed this effect. Both TNF-α and IFN-γ significantly reduced TER and increased epithelial permeability, effects prevented by ST/LA or BT. A Janus kinase (JAK) inhibitor synergistically potentiated effects of ST/LA or BT on TER and permeability, but p38, ERK1, 2, or PI3K inhibition did not. Finally, only probiotic-treated epithelial cells exposed to cytokines showed reduced activation of SOCS3 and STAT1,3. Conclusions: Deleterious effects of TNF-α and IFN-γ on epithelial function are prevented by probiotic, and to a lesser extent, commensal pretreatment. These data extend the spectrum of effects of such bacteria on intestinal epithelial function and may justify their use in inflammatory disorders. Background & Aims: Commensal bacteria are crucial for the development of the mucosal immune system. Probiotics are commensals with special characteristics and may protect mucosal surfaces against pathogens. Pathogens cause significant phenotypic alterations in infected epithelial cells, and probiotics reverse these deleterious responses. We hypothesized that probiotics and/or commensals may also reverse epithelial damage produced by cytokines. Methods: Human intestinal epithelial cells were exposed basolaterally to interferon (IFN)-γ (103 U/mL) or tumor necrosis factor (TNF)-α (10 ng/mL) for up to 48 hours and assessed for ion transport, transepithelial resistance (TER), and epithelial permeability in the presence or absence of probiotics (Streptococcus thermophilus [ST] and Lactobacillus acidophilus [LA]), or the commensal, Bacteroides thetaiotaomicron (BT). Results: Agonist-stimulated chloride secretion was inhibited by IFN-γ, an effect prevented by ST/LA or BT. The ability of ST/LA or BT to restore Cl− secretion was blocked by inhibitors of p38 MAPK, ERK1, 2, and PI3K. The cystic fibrosis transmembrane conductance regulator (CFTR) and the NKCC1 cotransporter were down-regulated by IFN-γ, and ST/LA pretreatment reversed this effect. Both TNF-α and IFN-γ significantly reduced TER and increased epithelial permeability, effects prevented by ST/LA or BT. A Janus kinase (JAK) inhibitor synergistically potentiated effects of ST/LA or BT on TER and permeability, but p38, ERK1, 2, or PI3K inhibition did not. Finally, only probiotic-treated epithelial cells exposed to cytokines showed reduced activation of SOCS3 and STAT1,3. Conclusions: Deleterious effects of TNF-α and IFN-γ on epithelial function are prevented by probiotic, and to a lesser extent, commensal pretreatment. These data extend the spectrum of effects of such bacteria on intestinal epithelial function and may justify their use in inflammatory disorders. Chronic inflammatory bowel disease (IBD) is believed to result from abnormal immune responses to the enteric microbial environment. The precise identity of the bacterial stimuli that cause IBD remains unclear. However, studies of experimental colitis in various animal models have shown the importance of the resident luminal flora in the initiation and perpetuation of intestinal inflammation. The colitis that develops spontaneously in numerous animal models1Lu J. Wang A. Ansari S. Hershberg R.M. McKay D.M. Colonic bacterial superantigens evoke an inflammatory response and exaggerate disease in mice recovering from colitis.Gastroenterology. 2003; 125: 1785-1795Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 2Hata K. Andoh A. Sato H. Araki Y. Tanaka M. Tsujikawa T. Fujiyama Y. Bamba T. 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Intestinal bacteria and ulcerative colitis.Curr Issues Intest Microbiol. 2003; 4: 9-20PubMed Google Scholar Several studies have shown a decreased number of lactic acid bacilli in patients with active Crohn's disease or ulcerative colitis, and increased numbers of Clostridia and Enterobacteriaceae.8Guarner F. Casellas F. Borruel N. Antolin M. Videla S. Vilaseca J. Malagelada J.R. Role of microecology in chronic inflammatory bowel diseases.Eur J Clin Nutr. 2002; 56: S34-S38Crossref PubMed Scopus (52) Google Scholar Taken together, these results, while indicating the critical role of intestinal luminal bacteria in triggering and maintaining chronic colitis, suggest a possible role for the manipulation of the bacterial flora to correct and/or prevent pro-inflammatory signaling in IBD.9Cummings J.H. Macfarlane G.T. Macfarlane S. Intestinal bacteria and ulcerative colitis.Curr Issues Intest Microbiol. 2003; 4: 9-20PubMed Google Scholar, 10Schölmerich J. Huber G. Biological therapy in IBD. Anti-tumor necrosis factor-alpha and others.Dig Dis. 2003; 21: 180-191Crossref PubMed Scopus (11) Google Scholar In addition to alterations in the microbiota, another common feature of IBD is increased cytokine production. Inflammation results from the action of pro-inflammatory cytokines. The intracellular signal transduction pathways of inflammatory cytokines have been studied extensively, and these pathways ultimately activate transcription factors, such as NFκB, Smads, and signal transducer and activator of transcription (STAT). I-κB, an NFκB negative-feedback regulator, is one of the major target genes of NFκB.11Sartor R.B. Sandborn W. Kirsner's inflammatory bowel disease. Saunders, Philadelphia2003Google Scholar Its expression is strongly induced by tumor necrosis factor (TNF)-α, interleukin 1 (IL-1), and lipopolysaccharide (LPS) in many cell types and leads to altered transcription of genes that contribute to the inflammatory process.11Sartor R.B. Sandborn W. Kirsner's inflammatory bowel disease. Saunders, Philadelphia2003Google Scholar, 12Ceponis P.J. McKay D.M. Ching J.C. Pereira P. Sherman P.M. Enterohemorrhagic Escherichia coli O157:H7 disrupts Stat1-mediated gamma interferon signal transduction in epithelial cells.Infect Immun. 2003; 71: 1396-1404Crossref PubMed Scopus (40) Google Scholar Likewise, other cytokine signals are transduced by the JAK/STAT pathway and these are regulated, in part, by suppressors of cytokine signaling (SOCS), which are endogenous JAK inhibitor proteins. Interestingly, several cytokines, including interferon (IFN)-γ and TNF-α, induce SOCS proteins, which serve as a feedback mechanism to downregulate cytokine signaling.13Krebs D.L. Hilton D.J. SOCS proteins negative regulators of cytokine signaling.Stem Cells. 2001; 19: 378-389Crossref PubMed Scopus (657) Google Scholar Dysregulation of SOCS-3 has been associated with active inflammation in mice models of colitis and arthritis.14Shouda T. Yoshida T. Hanada T. Wakioka T. Oishi M. Miyoshi K. Komiya S. Kosai K. Hanakawa Y. Hashimoto K. Nagata K. Yoshuimura A. Induction of the cytokine signal regulator SOCS3/CIS3 as a therapeutic strategy for treating inflammatory arthritis.J Clin Invest. 2001; 108: 1781-1793Crossref PubMed Scopus (337) Google Scholar, 15Suzuki A. Hanada T. Mitsuyama K. Yoshida T. Kamizono S. Hoshino T. Kubo M. Yamashita A. Okabe M. Takeda K. Akira S. Matsumoto S. Toyonaga A. Sata M. Yoshimura A. CIS3/SOCS3/SSI3 plays a negative regulatory role in STAT3 activation and intestinal inflammation.J Exp Med. 2001; 193: 471-484Crossref PubMed Scopus (422) Google Scholar Moreover, expression of a dominant-negative form of SOCS-3 in mice rendered them more susceptible to dextran sodium sulfate-induced colitis.15Suzuki A. Hanada T. Mitsuyama K. Yoshida T. Kamizono S. Hoshino T. Kubo M. Yamashita A. Okabe M. Takeda K. Akira S. Matsumoto S. Toyonaga A. Sata M. Yoshimura A. CIS3/SOCS3/SSI3 plays a negative regulatory role in STAT3 activation and intestinal inflammation.J Exp Med. 2001; 193: 471-484Crossref PubMed Scopus (422) Google Scholar There is increasing evidence that probiotic species can play a role in the treatment of IBD and other gastrointestinal disorders characterized by dysbiosis, namely, an altered intestinal microbiota.14Shouda T. Yoshida T. Hanada T. Wakioka T. Oishi M. Miyoshi K. Komiya S. Kosai K. Hanakawa Y. Hashimoto K. Nagata K. Yoshuimura A. Induction of the cytokine signal regulator SOCS3/CIS3 as a therapeutic strategy for treating inflammatory arthritis.J Clin Invest. 2001; 108: 1781-1793Crossref PubMed Scopus (337) Google Scholar Probiotics are "live microorganisms that survive passage through the gastrointestinal tract and have beneficial effects on the host."10Schölmerich J. Huber G. Biological therapy in IBD. Anti-tumor necrosis factor-alpha and others.Dig Dis. 2003; 21: 180-191Crossref PubMed Scopus (11) Google Scholar We have previously shown that deleterious functional effects induced in intestinal epithelial cells by enteroinvasive pathogens can be reversed by probiotic and/or commensal treatment.16Resta-Lenert S. Barrett K.E. Live probiotics protect intestinal epithelial cells from the effects of infection with enteroinvasive Escherichia coli (EIEC).Gut. 2003; 52: 988-997Crossref PubMed Scopus (503) Google Scholar, 17Resta-Lenert S. Barrett K.E. Enteroinvasive bacteria alter barrier and transport properties of human intestinal epithelium role of iNOS and COX-2.Gastroenterology. 2002; 122: 1070-1087Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar We and others have also shown that infection with enteric pathogens elicits marked pro-inflammatory responses in the gut mucosa.1Lu J. Wang A. Ansari S. Hershberg R.M. McKay D.M. Colonic bacterial superantigens evoke an inflammatory response and exaggerate disease in mice recovering from colitis.Gastroenterology. 2003; 125: 1785-1795Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 11Sartor R.B. Sandborn W. Kirsner's inflammatory bowel disease. Saunders, Philadelphia2003Google Scholar, 12Ceponis P.J. McKay D.M. Ching J.C. Pereira P. Sherman P.M. Enterohemorrhagic Escherichia coli O157:H7 disrupts Stat1-mediated gamma interferon signal transduction in epithelial cells.Infect Immun. 2003; 71: 1396-1404Crossref PubMed Scopus (40) Google Scholar, 17Resta-Lenert S. Barrett K.E. Enteroinvasive bacteria alter barrier and transport properties of human intestinal epithelium role of iNOS and COX-2.Gastroenterology. 2002; 122: 1070-1087Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar We hypothesized that probiotics and/or commensals may reverse epithelial damage produced by pro-inflammatory cytokines. This study therefore investigated the effect of probiotic and commensal bacteria on IFN-γ– and TNF-α–induced epithelial dysfunction. These studies used the human intestinal epithelial cell lines HT29/cl.19A and Caco-2, cultured as previously described.17Resta-Lenert S. Barrett K.E. Enteroinvasive bacteria alter barrier and transport properties of human intestinal epithelium role of iNOS and COX-2.Gastroenterology. 2002; 122: 1070-1087Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar Cells grown as polarized monolayers on HA Millicell filter inserts (12- and 24-mm diameter; Millipore, Bedford, MA) were used consistently within 14 days from seeding, or within 5 days postconfluency. Data obtained with both cell lines were qualitatively identical and have been used interchangeably in the results section. Probiotics, Streptococcus thermophilus (ST, ATCC19258) and Lactobacillus acidophilus (LA, ATCC4356), or a commensal, Bacteroides thetaiotaomicron (BT, ATCC29184) were cultured in deMane-Rogosa-Shape broths and agar (Difco Labs, Detroit, MI; ST and LA) or brain–heart infusion (Becton Dickinson, BBL, Sparks, MD; BT), respectively, and incubated overnight at 37°C in an anaerobic chamber. Bacterial cell suspensions were added apically to epithelial cell monolayers at a multiplicity of infection of 10:1 and incubated at 37°C for up to 48 hours. The experimental design included pretreatment with ST/LA or BT 1 hour before addition of the cytokines, or simultaneous addition of bacteria and cytokines. Because the 2 probiotic strains, ST and LA, were shown to act synergistically and to have additive effects in our system,16Resta-Lenert S. Barrett K.E. Live probiotics protect intestinal epithelial cells from the effects of infection with enteroinvasive Escherichia coli (EIEC).Gut. 2003; 52: 988-997Crossref PubMed Scopus (503) Google Scholar we present throughout the results of both strains used in combination. Some experiments were performed using probiotics that had been heat inactivated (1 hour at 100°C; ST/LAh).16Resta-Lenert S. Barrett K.E. Live probiotics protect intestinal epithelial cells from the effects of infection with enteroinvasive Escherichia coli (EIEC).Gut. 2003; 52: 988-997Crossref PubMed Scopus (503) Google Scholar Spent, centrifuged, and filtered (0.2 μmol) medium from ST/LA cultures (ST/LA-SM; corresponding to 7 × 108 CFU/mL), prepared as previously described,13Krebs D.L. Hilton D.J. SOCS proteins negative regulators of cytokine signaling.Stem Cells. 2001; 19: 378-389Crossref PubMed Scopus (657) Google Scholar or DNA extracted from stationary phase ST/LA18Flamm R.K. Hinrichs D.J. Thomashow M.F. Introduction of pAMb1 into Listeria monocytogenes by conjugation and homology between native L. monocytogenes plasmids.Infect Immun. 1984; 44: 157-161Crossref PubMed Google Scholar were also used in some experiments. DNA samples were resuspended at a final concentration of 25 μg/mL in ultrapure water (endotoxin, RNAse, and DNAse free). IFN-γ (103 U/mL) or TNF-α (10 ng/mL) were added to the basolateral side of cell monolayers and incubated for up to 48 hours before harvest. Pharmacologic inhibitors were used in some experiments (U0126, a MEK1 and 2 inhibitor, 30 μmol/L; SB203580, a p38 inhibitor, 1 μmol/L; tyrphostin AG490, a JAK2 inhibitor, 50 μmol/L; LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, 10 μmol/L; and SP600125, a JNK inhibitor, 10 μmol/L). Cell monolayers were tested for chloride secretion in modified Ussing chambers as described previously.17Resta-Lenert S. Barrett K.E. Enteroinvasive bacteria alter barrier and transport properties of human intestinal epithelium role of iNOS and COX-2.Gastroenterology. 2002; 122: 1070-1087Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar The mucosal and serosal baths contained Ringer's solution (composition in mmol/L: NaCl 115; NaHCO3 25; KH2PO4 0.4; K2HPO4 2.4; MgCl2 1.2; CaCl2 1.2; glucose 10; pH 7.4), gassed with 5% CO2–95% O2 and maintained at 37°C. The monolayers were continuously short-circuited by application of short circuit current (Isc); open circuit potential difference was measured every 1–5 minutes; conductance was calculated using Ohm's law. In other experiments, electrical resistance across infected and control monolayers was measured at various times using a "chopstick" voltohmeter (WPI, Sarasota, FL) with the inserts maintained at constant temperature (37°C) with 5% CO2 gassing. Measurements were expressed in Ω · cm2 after subtracting mean values of resistance obtained from cell-free inserts. Polarized cell monolayers were pretreated with probiotics for 1 hour, treated with cytokines as described, and harvested after 48 hours. Untreated cells were used as controls in all experiments. Fluorescein sulfonic acid (FS; Molecular Probes, Eugene, OR; MW= 478 Da, 200 μg/mL) or fluorescein isothiocyanate-dextran-10S (FITC-D10, Sigma, St. Louis, MO; MW = 10,000, 20 mg/mL) probes were added to the basolateral side.16Resta-Lenert S. Barrett K.E. Live probiotics protect intestinal epithelial cells from the effects of infection with enteroinvasive Escherichia coli (EIEC).Gut. 2003; 52: 988-997Crossref PubMed Scopus (503) Google Scholar The dishes were incubated at 37°C in a 5% CO2 incubator for 1 hour with gentle mixing at 15-minute intervals. After the initial incubation, at 15-minute intervals for up to 2 hours thereafter, the FS and FITC-D10 content of apical (Ap) and basolateral (Bl) samples was measured as the fluorescence intensity of 1:250 dilutions in distilled water (492-nm excitation and 515-nm emission wavelengths). Monolayer permeability was expressed as FS or FITC-D10 clearance: [probe]Bl − [probe]Ap/[probe]Bl, corrected for insert area (nL/cm2/h). Treated and untreated cells were harvested at various time points and processed as described.16Resta-Lenert S. Barrett K.E. Live probiotics protect intestinal epithelial cells from the effects of infection with enteroinvasive Escherichia coli (EIEC).Gut. 2003; 52: 988-997Crossref PubMed Scopus (503) Google Scholar, 17Resta-Lenert S. Barrett K.E. Enteroinvasive bacteria alter barrier and transport properties of human intestinal epithelium role of iNOS and COX-2.Gastroenterology. 2002; 122: 1070-1087Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar Proteins of interest were immunoprecipitated by overnight incubation at 4°C on a mixer with an appropriate dilution of specific antibody (anti-CFTR, anti-NKCC1, anti-SOCS3, anti-STAT3, anti-STAT1, anti-ERK1,2, anti-p38, anti-Akt, anti-JNK, anti- IκB-α from Santa Cruz Biotechnology, Santa Cruz, CA; anti-phosphotyrosine, anti-phosphoserine and anti-actin from Transduction Laboratories, Lexington, KY; anti-P-ERK1,2, anti-P-p38, anti-P-JNK from Upstate Biotechnology, Charlottesville, VA) in cold lysis buffer17Resta-Lenert S. Barrett K.E. Enteroinvasive bacteria alter barrier and transport properties of human intestinal epithelium role of iNOS and COX-2.Gastroenterology. 2002; 122: 1070-1087Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar supplemented with additional phenylmethylsulfonyl fluoride (20 μmol/L). The samples were then incubated with protein A agarose at 4°C for 1 hour with constant mixing. After washing, the immunoprecipitated proteins were electrophoresed through 7.5% or 4.5-15% polyacrylamide gels (BioRad, Hercules, CA) and transferred onto blotting membranes (Polyscreen PVDF, NEN, Boston, MA). After overnight blocking (PBS/Tween supplemented with 1% nonfat dry milk), blots were incubated with primary and secondary antibodies (HRP-conjugated anti-mouse IgG or anti-rabbit IgG, or anti-phosphotyrosine as appropriate) for 60 minutes at room temperature. Proteins were visualized by chemiluminescence (ECL Plus, Amersham, Piscataway, NJ) and exposed to X-OMAT film (Eastman Kodak, Rochester, NY). Total RNA was isolated from intestinal epithelial cells with an RNeasy kit (Quiagen, Valencia, CA) according to the manufacturer's instructions. RT-PCR was used to determine the expression of mRNA for SOCS3. β-Actin was used as an internal standard. Ten micrograms of RNA were separated on a 1% formaldehyde agarose gel and transferred to a GSP membrane (NEN Life Science Products). The membranes were then incubated with 32P-cDNA specific for SOCS3. The following primers were used to generate the cDNA: 5′-TCC CCT CGC CAC CTA CTG A-3′ and 5′-GGT CCA GGA ACT CCC GAA TG-3′.19Gatto L. Berlato C. Poli V. Tininini S. Kinjyo I. Yoshimura A. Cassatella M.A. Bazzoni F. Analysis of SOCS-3 promoter responses to interferon gamma.J Biol Chem. 2004; 279: 13746-13754Crossref PubMed Scopus (59) Google Scholar, 20Yan F. Polk B. Probiotic bacterium prevents cytokine-induced apoptosis in intestinal epithelial cells.J Biol Chem. 2002; 277: 50959-50965Crossref PubMed Scopus (443) Google Scholar The autoradiographs were quantified by densitometry scanning using BioRad MultiAnalist software (BioRad Laboratories). Unless otherwise indicated, all chemicals were of reagent grade and were obtained from Sigma (St. Louis, MO). All data are expressed as means ± SEM. Statistical analysis was performed by repeated measures ANOVA and P values < .05 were considered statistically significant. We investigated the effect of probiotics and a commensal on IFN-γ–induced alterations in ion transport. Simultaneous treatment of cell monolayers with bacteria plus cytokines failed to modulate cytokine effects and thus here we discuss only results based on bacterial pretreatment for 1 hour followed by IFN-γ (103 U/mL). Chloride secretion, measured as changes in short circuit current (Isc) induced by forskolin, was assessed at various times thereafter by mounting the monolayers in Ussing chambers. As expected, exposure of cells to IFN-γ for up to 48 hours caused a significant reduction in forskolin-stimulated chloride secretion (Figure 1A). However, pretreatment with either ST/LA, or BT protected against the effect of IFN-γ on chloride secretion. Heat-inactivated ST/LA, DNA extracted from ST/LA, or ST/LA spent medium were unable to reproduce the effect of live ST/LA (Figure 1B). Pharmacologic inhibitors were used to explore cellular signaling pathways involved in the effects of IFN-γ and the bacteria. The effect of IFN-γ alone on agonist-stimulated chloride secretion was unaffected by inhibition of MEK1,2 or JNK, whereas the inhibitory effect of the cytokine was reduced in the presence of a p38 inhibitor, and abolished by a JAK2 inhibitor (Figure 2A). Similar studies were used to assess how probiotics and commensals reversed the effect of IFN-γ on chloride secretion (Figure 2B and 2C). These experiments revealed that the ability of the probiotics and the commensal to reverse IFN-γ–induced alterations of ion transport are likely mediated through ERK1 and 2, p38, and PI3K. On the other hand, there was no evidence that JNK plays a role. Involvement of JAK in the protective effect of probiotics or the commensal could not be assessed because the JAK inhibitor alone prevented the effect of IFN-γ. Finally, in contrast to the effects of IFN-γ, TNF-α had no effect on forskolin-stimulated chloride secretion in HT29/cl.19A or Caco-2 cells at any time up to 48 hours.Figure 2The protective effect of probiotics on IFN-γ–dysregulated ion transport can be reversed in part by treatment with specific kinase inhibitors: HT29/cl.19A cell monolayers were treated with various kinase inhibitors (SB203580, 1 μmol/L; U0126, 30 μmol/L; SP600125, 10 μmol/L; AG490, 50 μmol/L; LY294002, 10 μmol/L) for 15 minutes and then with bacteria or medium alone for 1 h prior to addition of IFN-γ (103 U/mL). Chloride secretion, measured as changes in short circuit current (Isc) in response to Bl addition of forskolin (10 μmol/L), was tested at various times after treatment in Ussing chamber-mounted monolayers. The panels show the effects of the pharmacologic treatments in cells exposed to IFN-γ alone (A), ST/LA and IFN-γ (B), or BT and IFN-γ (C). Note that IFN-γ was present under all conditions studied, except the data labeled "control" in (A). Results are expressed as means ± SEM, N = 6. Statistical analysis was by ANOVA (*,#, P < .05; **, P < .01; ***, P < .001. (A) Conditions are compared to the untreated control. (B and C) Conditions are compared to the respective controls in the presence of IFN-γ, and in the absence (*) or presence (#) of inhibitors. Where error bars are not shown, they were obscured by the symbols.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Others have shown that the effect of IFN-γ on chloride secretion may be explained, at least in part, by a reduction in the expression of membrane transporters.21Yoo D. Lo W. Goodman S. Ali W. Semrad C. Field M. Interferon-gamma downregulates ion transport in murine small intestine cultured in vitro.Am J Physiol Gastrointest Liver Physiol. 2000; 279: G1323-G1332PubMed Google Scholar We therefore assessed whether probiotics or a commensal could prevent these changes. Cell monolayers were exposed to ST/LA or BT for 1 hour and then treated with IFN-γ. Cells were harvested at 48 hours and, after lysis, analyzed for CFTR and NKCC1 content by Western blotting (Figure 3A and 3B). IFN-γ reduces both CFTR and NKCC1 levels. ST/LA fully reversed the effect of the cytokine, whereas the effect of BT was only partial. Overall, these results support the hypothesis that live probiotics or, to a lesser extent, commensal bacteria, preserve epithelial transport function in the face of inflammation by modulating protein expression. We next examined effects of bacteria on cytokine-induced changes in epithelial barrier function. Figure 4 shows that treatment of cell monolayers with either IFN-γ or TNF-α for 48 hours resulted in a significant decrease in TER, as expected.22Schultz M. Strauch U.G. Linde H.J. Watzl S. Obermeier F. Gottl C. Dunger N. Grunwald N. Scholmerich J. Rath H.C. Preventive effects of Escherichia coli strain Nissle 1917 on acute and chronic intestinal inflammation in two different murine models of colitis.Clin Diagn Lab Immunol. 2004; 11: 372-378PubMed Google Scholar However, ST/LA or BT pretreatment reversed IFN-γ– (Figure 4A) or TNF-α–induced (Figure 4B) decreases in TER. Simultaneous treatment with ST/LA or BT (data not shown), or pretreatment with heat-inactivated ST/LA, ST/LA-SM, or ST/LA-DNA did not prevent the effect of IFN-γ or TNF-α on TER. In studies designed to identify the mechanisms of these effects, the ability of IFN-γ alone to reduce TER was reversed by a JAK inhibitor but not by inhibition of ERK, p38, PI3K, or JNK (Figure 5A). These data underscore the fact that different intracellular signaling pathways regulate secretory responses versus barrier function. In the presence of probiotics or a commensal, reversal of the effects of IFN-γ on TER was abrogated in part by pharmacologic inhibition of ERK, p38, and PI3K, but not of JNK (Figure 5B and C). Again, because the JAK inhibitor reversed the effect of IFN-γ alone, the role of JAK in probiotic effects could not be tested.Figure 5The protective effect of probiotics on barrier function can be reversed, in part, by treatment with specific kinase inhibitors. Caco-2 cell monolayers were treated with kinase inhibitors (SB203580, 1 μmol/L; U0126, 30 μmol/L; SP600125, 10 μmol/L; AG490, 50 μmol/L; LY294002, 10 μmol/L) for 15 minutes and then with bacteria or medium alone for 1 hour prior to addition of IFN-γ (103 U/mL). Transepithelial resistance (TER) was measured 48 hours after treatment using a volthometer. The figure shows data for cell monolayers exposed to IFN-γ in the absence of bacteria (A), cell monolayers treated with probiotics and IFN-γ (B), and cell monolayers treated with a commensal and IFN-γ (C). Results are expressed as means ± SEM, N = 6. Statis
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