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TRANSFORMING GROWTH FACTOR-"1 PROTECTS AGAINST INTESTINAL EPITHELIAL BARRIER DYSFUNCTION CAUSED BY HYPOXIA-REOXYGENATION Kathryn L. Howe,*† Robert J. Lorentz,* Amit Assa,* Lee J. Pinnell,* Kathene C. Johnson-Henry,* and Philip M. Sherman*‡ *Cell Biology Program, Research Institute, Hospital for Sick Children, University of Toronto, Toronto; †Faculty of Health Sciences, Department of Surgery, Division of General Surgery, McMaster University, Hamilton, Ontario; and ‡Department of Paediatrics, Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada Received 3 Sep 2014; first review completed 22 Sep 2014; accepted in final form 5 Jan 2015 ABSTRACT—Intestinal epithelia regulate barrier integrity when challenged by inflammation, oxidative stress, and microbes. Transforming growth factor-"1 (TGF-"1) is a cytokine with known beneficial effects on intestinal epithelia, including barrier enhancement, after exposure to proinflammatory cytokines and infectious agents. The aim of this study was to determine whether TGF-"1 directly protects intestinal epithelia during hypoxia-reoxygenation (HR). Intestinal epithelial monolayers (T84, Caco-2bbe) were exposed to either hypoxia (1% O2, 1 h) or oxidative stress (hydrogen peroxide, 1 mM), followed by normoxic atmosphere for different time points in the absence and presence of varying concentrations of TGF-"1. Transepithelial electrical resistance (TER) assessed barrier function, with RNA extracted for reverse transcription polymerase chain reaction analysis of GPx-1, HIF-1, heme-oxygenase-1 (HO-1), and NOX-1. In some experiments, intestinal epithelia were exposed to enterohemorrhagic Escherichia coli (EHEC) O157:H7 during the reoxygenation period and TER recorded 7 h after the infectious challenge. Hypoxia-reoxygenation significantly decreased TER in intestinal epithelia compared with normoxic controls. Transforming growth factor-"1 pretreatment ameliorated HR-induced epithelial barrier dysfunction in T84 (at 1 Y 3 h) and Caco-2bbe (1 h) monolayers. Transforming growth factor-"1 preserved barrier integrity for up to 16 h after challenge with hydrogen peroxide. In TGF-"1Ytreated epithelial monolayers, only HO-1 mRNA significantly increased after HR (P G 0.05 vs. normoxic controls). The EHEC-induced epithelial barrier dysfunction was significantly worsened by intestinal HR (P G 0.05 vs. normoxia-EHECYinfected cells), but this was not protected by TGF-"1 pretreatment. Transforming growth factor-"1 preserves loss of epithelial barrier integrity caused by the stress of HR via a mechanism that may involve the upregulation of HO-1 transcription. Targeted treatment with TGF-" could lead to novel therapies in enteric diseases characterized by HR injury. KEYWORDS—Ischemia-reperfusion, intestinal permeability, cytokine, antioxidant
INTRODUCTION
enterohemorrhagic Escherichia coli (EHEC) O157:H7 (4). Transforming growth factor-" exerts protective effects in a murine model of colitis (5) and in several models of I/R injury, including myocardial infarction (6), stroke (7), and renal damage (8). Indirect evidence suggests that TGF-" also is protective in the setting of intestinal ischemia (9). Clinical studies demonstrate that TGF-" signaling is downregulated in IBD, a condition of inflammatory hypoxia, with SMAD7 antisense oligonucleotide treatment improving clinical signs and symptoms of active Crohn disease (10). Despite a common underlying mechanism for many of these conditions, it remains to be determined whether TGF-" directly protects intestinal epithelia in models of hypoxia and I/R. Given the properties of TGF-", we sought to determine using an epithelial cell culture model system whether 1) TGF- " protects against hypoxia-reoxygenation (HR)Yinduced intestinal epithelial barrier dysfunction, 2) HR exacerbates barrier dysfunction caused by a pathogenic bacterium, and 3) TGF-" protects against a double hit of exposure to both HR and an enteric pathogen.
Hypoxic conditions underlie the pathobiology of a variety of gastrointestinal conditions, including chronic inflammatory bowel diseases (IBDs), enteric infections, necrotizing enterocolitis, and ischemia-reperfusion (I/R) injury caused by mesenteric ischemia and organ transplantation (1). The epithelium lining the intestinal tract is composed of a highly metabolically active single layer of cells with both absorptive and barrier functions that exist in a state of physiologic hypoxia (2). The functional integrity of gut barrier function can be altered when challenged by proinflammatory cytokines, immune cells, oxidative stress, and pathogenic microbes. Transforming growth factor-" (TGF-") is an antiinflammatory cytokine with beneficial effects on intestinal epithelial barrier function, including barrier enhancement and protection against dysfunction caused by proinflammatory cytokines and infections, such as Cryptosporidium parvum (3) and Address reprint requests to Philip M. Sherman, MD, FRCPC, Peter Gilgan Centre for Research and Learning, 686 Bay St, Room 21-9430 Toronto, Ontario, Canada M5G 0A4. E-mail: [email protected]. This study was supported by the Canadian Institutes for Health Research (grants MOP-89894 and IOP-92890 to P.M.S.). P.M.S. is a recipient of the Canada Research Chair in Gastrointestinal Disease. None of the authors have any relevant conflicts of interest to report. DOI: 10.1097/SHK.0000000000000333 Copyright Ó 2015 by the Shock Society
METHODS Cell and bacterial culture T84 intestinal epithelial cells purchased from the American Type Culture Collection (Manassas, Va) were grown and maintained in a 1:1 (vol:vol) mixture of Dulbecco modified Eagle medium and Ham F-12 medium containing L-glutamine (0.365 g/L) and sodium bicarbonate (2.438 g/L), supplemented with 483
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FIG. 1. Transforming growth factor-"1, but not TGF- " 2, increases TER of polarized intestinal epithelia. Transforming growth factor-"1 (0.1 Y 100 ng/mL) added to the basolateral wells of either T84 (A) or Caco-2 (panel B) monolayers and TER was recorded after 24, 48, and 72 h. Values are expressed as percentage of control monolayers (A, n = 8 monolayers in four separate experiments [control TER 1,966 T 331, 2,145 T 435, and 1,899 T 490 4 & 0.33 cm2 for 24, 48, and 72 h, respectively). B: w/o denotes washout after 24 h, 10 ng/mL; n = 5 Y 11 monolayers in four experiments. #P G 0.05, *P G 0.01, **P G 0.001 vs. control monolayers (control TER 637 T 51, 548 T 51, and 520 T 53 4 & 0.33 cm2 for 24, 48, and 72 h, respectively). Transforming growth factor-"2 (10 ng/mL) was added to the basolateral well of T84 (C) or Caco-2 (D) monolayers, and TER was measured at 24-h intervals for up to 3 days. Values are expressed as percentage control monolayer TER. C, n = 9 monolayers in four experiments (control TER 2,263 T 609, 1,952 T 388, and 1,592 T 296 4 & 0.33 cm2 for 24, 48, and 72 h, respectively). D, n = 6 monolayers used in two experiments (control TER 859 T 50, 699 T 37, and 632 T 40 4 & 0.33 cm2 for 24, 48, and 72 h, respectively). P = N.S. versus control and between groups (ANOVA).
fetal bovine serum (10%) and 2% penicillin-streptomycin at 37-C in 5% CO2 (all from Invitrogen, Burlington, Ontario, Canada). Caco-2bbe (American Type Culture Collection, labeled Caco-2 hereafter) cells were grown and maintained in Dulbecco modified Eagle medium containing L-glutamine (0.584 g/L) and supplemented with 10% fetal bovine serum, 2% penicillin-streptomycin, sodium pyruvate (1 mM), and human transferrin (0.01 mg/mL) at 37-C in 5% CO2 (all from Invitrogen). Recombinant human TGF-"1 (TGF-" throughout text, unless otherwise specified), TGF-"2, or latent TGF-" (R&D Systems Inc, Minneapolis, Minn) were added into the basolateral side of wells (0.1 Y 100 ng/mL, 1 Y 72 h), and transepithelial electrical resistance (TER) was recorded throughout the experimental period. Enterohemorrhagic E. coli O157:H7 strain CL56 (11) was maintained on 5% sheep blood agar plates (Oxiod, Nepean, Ontario, Canada) at 4-C, cultured overnight in nonaerated Luria-Bertani broth at 37-C, and then added to the apical compartment of intestinal epithelia at approximately 1 108 colonyforming units/monolayer.
experiment, cells were placed in either warmed normoxic or hypoxic tissue culture medium for 60 min, TER was recorded (time 0), and reoxygenation was established by replacing each well with normoxic medium for up to 24 h. The TER was recorded at 1, 2, 3, and 24 h. In experiments testing the effect of TGF-", a single dose (10 ng/mL in normoxic culture medium) was added into the basolateral compartment 60 min before hypoxic challenge. For experiments determining whether HR exacerbates EHEC O157:H7Yinduced barrier dysfunction, epithelial monolayers were inoculated at the start of reoxygenation (time 0), and TER was recorded at 7 h after infection.
Oxidative stress T84 and Caco-2 cells were washed once in supplement-free culture medium and equilibrated for 1 h at 37-C in the absence or presence of TGF-" (10 ng/mL). Hydrogen peroxide (30% wt/wt; Sigma-Aldrich) was added to the apical surface of T84 or Caco-2 monolayers (50 2M Y 5 mM), and TER was recorded (multiple time points were measured up to 16 h).
Gene expression
Epithelial permeability Caco-2 and T84 cells were seeded onto the apical surface of semipermeable filter supports (surface area, 0.33 cm2 and 1 cm2, respectively; Corning Inc, Corning, NY) and cultured until the TER (measured with a Voltmeter and chopstick electrodes, Millicell-ERS; Millipore, Bedford, Mass) was greater than 1,000 4 cm2, which is indicative of tight junction formation. Changes in TER during the experimental protocol are indicative of alterations in epithelial barrier function. Paracellular permeability was also tested using a nonabsorbable fluoresceinisothiocyante (FITC)Ylabeled Dextran probe (10 kd; Sigma-Aldrich, St. Louis, Mo), as previously described (12). Briefly, FITC-Dextran (1 mg/mL in cell culture medium) was applied into the apical compartment of wells, and recovery of the probe in the basolateral side was measured after 1 h. Fluorescent units were calculated using a microplate fluorescence reader (Victor, Perkin-Elmer, Mass), and results were expressed as optical density arbitrary units.
Hypoxia-reoxygenation Hypoxic conditions were generated using a sealed modular chamber and flow meter system (Billups-Rothenberg Inc, Del Mar, Calif) flushed at 20 L/ min (5 min) with filtered 1% O2 (5% CO2, balance N2; Praxair, Danbury, Conn) (13). For hypoxic conditions, cell culture medium was placed into a vented T25 culture flask (Corning) and flushed with 1% O2, and the sealed chamber was placed in an incubator overnight at 37-C. At the start of an
Total RNA was extracted from cells grown on monolayers at the completion of experimental conditions using TRIzol Isolation Reagent, as per manufacturer’s instructions (Life Technologies, Burlington, Ontario, Canada). For HR experiments, samples were collected at 1 h after HR, whereas HRbacteria experiments were collected at 7 h after HR-EHEC challenge. Only samples with A260/280 more than 1.8 were used to synthesize cDNA using the iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, Calif). Primers were as follows: HIF-1!: forward, 5¶-TCACCACAGGACAG TACAGGATGC-3¶; reverse, 5¶-CCAGCAAAGTTAAAGCATCAGGTTCC-3¶ (14) GPx-1: forward, 5¶-CC AAGCTCATCACCTGGTCT-3¶; reverse, 5¶-TCGAT GTCAATGGTCTGGAA-3¶ (15) HO-1: forward, 5¶-AAGATTGCCCAGAAAGCCCTGGAC-3¶; reverse, 5¶-AACTGTCGCCACCAGAAAGCTGAG-3¶ (16) NOX-1: forward, 5¶-GGAGCAGGATTGGGGTCAC-3¶; reverse, 5¶TTGCTGTCCCATCCGGTGAG-3¶ (17) GAPDH: forward, 5¶-GTCATCCATGACAACTTTGG-3¶; reverse, 5¶-GAGCTTGACAAAGTGGTCGT-3¶ (14). Reverse transcription polymerase chain reaction was performed using SsoFast Eva-Green Supermix (Bio-Rad Laboratories) and CFX96 C1000 Thermal Cycler (Bio-Rad Laboratories). Values were normalized against the housekeeper gene GAPDH and then compared between experimental groups (18).
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FIG. 2. Hypoxia-reoxygenation decreases TER in intestinal monolayers. T84 (A) or Caco-2 (B) epithelial monolayers were exposed to either normoxia (open bars) or hypoxic conditions (1% O2, closed bar histograms) for 1 h, reoxygenated with normoxic tissue culture medium, and TER was recorded at 1, 2, 3, and 24 h. Values are expressed as TER (percentage of value at time 0) (A, n = 8 Y 15 monolayers used in three to six separate experiments; B, n = 8 Y 11 monolayers used in three to four experiments; #P G 0.05, *P G 0.01, **P G 0.001 vs. normoxic control [paired t test]). Note that, for ease of data legibility, the y axis originates at 80% of baseline TER.
Statistical analyses Data are presented as means T SEM. Data were analyzed using one-way analysis of variance (ANOVA), followed by post hoc comparisons using Tukey test, or using the paired Student t test, with P G 0.05 taken as the level of statistical significance.
RESULTS TGF-"1, but not TGF-"2, promotes epithelial barrier integrity
Using two intestinal epithelial cell lines, cryptlike T84 and villus-like Caco-2bbe, TGF-"1 (Fig. 1, A and B), but not TGF"2 (Fig. 1, C and D), enhanced barrier function in both a doseand a time-dependent manner. Accordingly, TGF-"1 was selected for use in all subsequent experiments (labeled hereafter as TGF-"). TGF-" preserves epithelial barrier dysfunction caused by HR
Using a modified HR model previously described by Xu et al. (13), HR significantly decreased intestinal epithelial barrier TER after 1, 2, 3, and 24 h of reoxygenation in T84 monolayers (Fig. 2A). As shown in Figure 3A, pretreatment with TGF-" or latent TGF-" ameliorated the effects of HR. Corroborating the findings of TER, HR also resulted in
increased paracellular permeability to a fluorescent-labeled macromolecular Dextran probe (Fig. 4). Using a complementary intestinal epithelium model, Caco2bbe (Caco-2) cells, we showed that the effects were not cell line specific. Hypoxia-reoxygenation also caused decreased TER of Caco-2 monlayers at 1 and 2 h after restoring normoxic conditions (Fig. 2B), an effect that was significantly ameliorated by TGF-" after 1 h of reoxygenation only (Fig. 3B). TGF-" ameliorates barrier dysfunction caused by H2O2
Hypoxia-reoxygenation produces downstream mediators including reactive oxygen species (ROS) known to cause barrier dysfunction (19, 20). Pretreatment with TGF-" for 1 h, but not the negative control, latent (inactive) TGF-", protected epithelial barrier dysfunction caused by H2O2 (1 mM) for up to 4 h in T84 (Fig. 5A) and at 3 and 16 h in Caco-2 monolayers (Fig. 5B). HR exacerbates epithelial barrier dysfunction caused by an enteric pathogen
In both T84 (Fig. 6A) and Caco-2 (Fig. 6B) monolayers, HR caused greater reductions in TER than observed with EHEC infection under conditions of normoxia.
FIG. 3. TGF-" improves HR-induced barrier dysfunction in intestinal epithelia. T84 (A) and Caco-2 (B) epithelial monolayers were treated with either TGF-"1 or latent TGF-" (10 ng/mL) for 1 h before HR challenge, and TER was then measured at 1, 2, 3, and 24 h. Values are expressed as the percentage of baseline TER at time 0. Normoxia alone is denoted in open bar histograms, normoxia plus TGF-"1 in crosshatch bar histograms, HR alone in closed bar histograms, HR + TGF-"1 in diamond bar histograms, and HR + latent TGF-" indicated by the horizontal line bar histograms (A, n = 6 Y 12 monolayers used in two to four experiments; B, n = 7 Y 9 monolayers tested in three separate experiments; #P G 0.05, *P G 0.01, **P G 0.001 vs. normoxic controls, lines above the bar histograms represent statistically significant differences between groups). For ease of data legibility, the y axis originates at 70% to 85% of baseline TER.
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FIG. 4. Hypoxia-reoxygenation increases paracellular permeability of polarized epithelia, which is ameliorated by TGF-". T84 monolayers were exposed to normoxia (left bar histogram) or hypoxia for 1 h (1% O2) either in the absence (middle bar histogram) or presence of TGF-"1 (10 ng/mL for 1 h) (right bar) followed by placing normoxic tissue culture medium containing FITCDextran (10 kd, 1 mg/mL) into the apical compartment. After 1 h, samples were collected from the basolateral compartment of polarized epithelia for optical density readings (n = 8 Y 10 monolayers in three to four experiments; *P G 0.01 vs. normoxia; line above the histograms represents a statistically significant difference between HR study groups).
TGF-"1 does not ameliorate the effect of HR on pathogen-induced barrier dysfunction
Pretreatment of T84 monolayers with TGF-" (10 ng/mL) for 1 h followed by HR and EHEC O157:H7 (7 h) did not prevent the drop in TER induced by the pathogen (54.6% T 4.9 % of baseline TER in the absence of TGF-" vs. 52.3% T 3.9% in the presence of TGF-"; n = 13 Y 14 monolayers tested in five separate experiments [data not shown]). TGF-" pretreatment increases expression of the antioxidant HO-1 in monolayers exposed to HR
To begin to elucidate mechanisms underlying TGF-" protection during HR, RNA was collected from intestinal epithelial monolayers. Levels of mRNA expression for GPx-1, NOX-1, and HIF1! were not significantly different between treatment groups (data not shown). By contrast, transcription of HO-1 was significantly altered by TGF-" treatment in gut monolayers exposed to HR (Fig. 7). DISCUSSION Intestinal ischemia and reperfusion injury is relevant to the pathophysiology of many gut diseases, with a common underlying
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process involving inflammatory mediators, epithelial barrier disruption, and bacterial translocation (21Y23). Proinflammatory cytokine mediators are upregulated in response to inflammatory or infectious insults in the intestine, which is balanced by concurrent upregulation of anti-inflammatory mediators that serve to quell the responses to injury. Transforming growth factor-" is a predominantly anti-inflammatory cytokine that regulates the function of many cells in the intestinal milieu, including epithelia, monocytes, and dendritic cells that interface with immune cells in the underlying lamina propria. Given the protective effect of TGF-" on intestinal epithelia (3, 4), we sought to determine if this antiinflammatory cytokine could ameliorate responses of intestinal epithelia to HR, an insult underlying many intestinal diseases. Studies using organ transplant, septic shock, and multiorgan failure models have advanced current understanding of mechanisms underlying I/R injury in the gut. Interest has more recently focused on the role of ongoing HR in chronic diseases, including IBDs (2). Importantly, the intestinal epithelium is uniquely able to withstand hypoxia through a mechanism involving upregulation of intestinal trefoil factor by HIF-1! (24). Models aimed at understanding these processes have begun to outline the inflammatory responses and barrier dysfunction caused not only by ischemia or hypoxia but also the ROS and intracellular signaling pathways generated in response (25). In this study, we continue to build on previous work by others (13, 15) showing that HR leads to epithelial barrier dysfunction after exposure of both cryptlike (T84) and villus-like (Caco-2bbe) intestinal cell lines and demonstrate that TGF-"1 pretreatment prevents HR-induced barrier dysfunction. Paracellular permeability studies corroborated these data, showing not only that HR increases permeability but also that TGF-" pretreatment for 1 h can prevent this effect. The protective effects of TGF-"1 on HR occurred at times that are well in advance of independent effects previously shown on barrier enhancement of the apical junction complexes, which are first observed after 16 h (4). Interestingly, the protective effect of a single hour-long pulse of TGF-" has been observed previously in a model of EHEC O157:H7Yinduced barrier dysfunction (4). In a murine model of chemical-induced colitis (Dextran sodium sulfate), recombinant TGF-" delivered to the gut protected against both acute and chronic injury (5), which likely reflects prevention of inflammation-induced HR. Importantly, phase I clinical trials have shown that restoration of
FIG. 5. TGF-" ameliorates disruption of TER caused by H2O2, an ROS produced by HR. T84 (A) and Caco-2 (B) monolayers were either left untreated (white bars) or pretreated with TGF-"1 (light gray bars) or latent TGF-" (1 h, 10 ng/mL, dark gray bars), equilibrated in plain medium (1 h), and subsequently treated with H2O2 (1 mM, apical well, black bars). Values are expressed as percentage of baseline TER (A, n = 3 Y 12 monolayers in up to three experiments; B, n = 4 monolayers used in two experiments; #P G 0.05, *P G 0.01 vs. normoxic controls, #*P G 0.01 vs. H2O2 and latent TGF-" + H2O2).
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FIG. 6. Hypoxia-reoxygenation exacerbates EHEC O157:H7Yinduced epithelial barrier dysfunction. T84 (A) and Caco-2 (B) polarized monolayers were exposed to hypoxic conditions (1% O2) for 1 h, reoxygenated with normoxic tissue culture medium in the absence or presence of EHEC O157:H7 (~1 108 colony-forming units/monolayer) placed into the apical compartment with TER measured 7 h after the infectious challenge. Values are expressed as percentage of TER measured at time 0 (A, n = 6 Y 15 monolayers used in three to five experiments, *P G 0.01, **P G 0.001, ***P G 0.0001, ANOVA, followed by paired Student t test versus normoxic controls; B, n = 6 monolayers in three experiments, **P G 0.01, paired Student t test).
TGF-" signaling in active Crohn disease is safe, well tolerated, and seems to improve disease activity (10). Our results add to this body of emerging data by showing that TGF-" is protective in gut HR. Reactive oxygen species are generated as a downstream event of HR and have direct damaging effects on the intestinal epithelium (26). Our study showed that pretreatment with TGF-" for 1 h prevented the barrier defect caused by direct H2O2 application to the T84 and Caco-2 cell lines. Although the preventive effect of TGF-" was statistically significant for up to 4 h in T84 monolayers, the continued trend persisted for several hours. In the Caco-2 monolayers, however, the effect of H2O2 was less robust and, notably, TGF-" was significantly protective at 3 and 16 h only. These data are compatible with those of Basuroy et al. (27) who demonstrated that epidermal growth factor prevents H2O2-induced barrier dysfunction in Caco-2 cells through direct action on the mitogen-activated protein kinase signaling pathway, ERK, and subsequent regulation of tight junction proteins and the actin cytoskeleton. The concentration of H2O2 (1 mM) we used is consistent with the optimal concentration determined by authors using the IPECJ2 small intestinal epithelial cell line (28). Interestingly, in contrast to findings using H2O2, latent TGF-" protected against HR-induced barrier dysfunction, despite its planned use as a negative control. Although activators of latent TGF-" in vitro are extreme pH, heat, and ionizing radiation, none of these were factors during the 60-min application of the latent cytokine. One possible explanation for the discrepancy is that, despite washout, residual latent TGF-" was activated during HR exposure, thereby releasing active TGF-" from the latency-associated peptide. Unlike the global effects of HR on cell monolayers, H2O2 was applied directly to the apical surface where contact with basolaterally applied latent TGF-" was physically excluded. Consequently, the inactive form of TGF-" was not protective against H2O2-induced epithelial barrier dysfunction. The HR-induced barrier dysfunction model was also used to test the effects of additional insults by exposing cell lines to EHEC O157:H7. Hypoxia-reoxygenation exacerbated the barrier defects caused by exposure to the microbe alone, findings consistent with a previous study demonstrating exposure to hypoxia and nonpathogenic E. coli followed by 3 h of normoxia caused permanent barrier disruption in rat mucosal ileal sections,
unlike the reversible effect caused by hypoxia alone (29). Although they used a putative nonpathogenic strain, we chose to test enteropathogenic bacteria because these predominate in settings of sepsis and other chronic inflammatory conditions where the diversity of the normal gut microbiota has been reduced by the use of antibiotics (30). Epithelia placed under pharmacologically induced metabolic stress also have compromised barrier function when exposed to commensal bacteria (31), supporting the hypothesis that stress exacerbates host-microbial interactions. Given the role of TGF-" in preserving epithelial barrier during I/R injury (9) and protecting against colitis in mice with genetically engineered barrier compromise (32), we hypothesized that pretreatment with TGF-" would protect against the double insult of HRYpathogenic bacteria. However, the data were not supportiveVsurprising given that TGF-" ameliorates epithelial barrier disruption caused by HR and the ROS product, H2O2, and that it prevents barrier defects caused by EHEC O157:H7 (4). It is possible that the virulence of pathogenic bacteria in the setting of HR may be too great an insult to overcome with TGF-" pretreatment. Indeed, recent studies have determined that HR causes intestinal epithelial release of adenosine and dynorphin and direct activation of the quorum sensing circuitry of Pseudomonas aeruginosa, leading to increased expression of virulence determinants and greater barrier dysfunction (33). This work has since been corroborated
FIG. 7. TGF-" increases epithelial HO-1 gene expression in the setting of HR. T84 monolayers were pretreated with TGF-"1 (10 ng/mL for 1 h) before challenge with HR. RNA was collected after 1 h of reoxygenation, and HO-1 mRNA expression was measured relative to the housekeeping gene GAPDH. n = 8 Y 12 monolayers tested in three separate experiments. Horizontal line above the bar histograms represents a difference between groups, *P G 0.05 by ANOVA.
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in vivo, where I/R causes 100% mortality in mice cochallenged with P. aeruginosa compared with less than 10% for bacterial challenge in the absence of I/R or less than 50% for I/R alone (34). In contrast to the HR-pathogen experiments, it is noteworthy that, despite the greater barrier insult caused by H2O2, TGF-" was protective for a period, suggesting the highly relevant contribution of microbes to the regulation of gut permeability. We show that HO-1 mRNA is upregulated at a time when TGF-" protective effects during HR are evident. This is consistent with previous reports indicating that HO-1 is a mediator of gastroprotective pathways (35) and that increased HO-1 expression protects against I/R injury in the intestine (36). More recently, in a mouse model of necrotizing enterocolitis, HO-1 deficiency was shown to promote the development of intestinal injury after oral challenge with formula and exposure to HR (37). Previous studies show that both curcumin and hirsutenone ameliorate oxidant-induced intestinal epithelial barrier disruption by upregulating HO-1 (20). Future studies, therefore, should determine whether limiting induction of HO-1(using either a chemical inhibitor or silencing RNA) blocks the protective effects of TGF-" in the setting of HR. Taken together, the findings in this report show that TGF-" protects against HR-induced intestinal barrier dysfunction, as well as that induced by the ROS H2O2. A mechanism for this beneficial effect may involve the upregulation of HO-1 specifically. By contrast, TGF-" was not able to protect against the exacerbated barrier dysfunction caused by HR and enteric bacterial pathogens. These studies emphasize the diverse impacts of HR on intestinal epithelia and propose a means that could be used, at least in some clinical settings, to ameliorate such effects. Targeted TGF-" therapy holds promise for use in managing intestinal disease processes characterized by HR.
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Xu DZ, Lu Q, Deitch EA: Nitric oxide directly impairs intestinal barrier function. Shock 17(2):139Y145, 2002. 24. Furuta GT, Turner JR, Taylor CT, Hershberg RM, Comerford K, Narravula S, Podolsky DK, Colgan SP: Hypoxia-inducible factor 1Ydependent induction of intestinal trefoil factor protects barrier function during hypoxia. J Exp Med 193(9):1027Y1034, 2001. 25. Elias-Miro M, Jimenez-Castro M, Rodes J, Peralta C: Current knowledge on oxidative stress in hepatic ischemia/reperfusion. Free Radic Res 47(8): 555Y568, 2013. 26. Tomita T, Sadakata H, Tamura M, Matsui H: Indomethacin-induced generation of reactive oxygen species leads to epithelial cell injury before the formation of intestinal lesions in mice. J Physiol Pharmacol 65(3):435Y440, 2014. 27. Basuroy S, Seth A, Elias B, Naren AP, Rao R: MAPK interacts with occludin and mediates EGF-induced prevention of tight junction disruption by hydrogen peroxide. Biochem J 393(Pt 1):69Y77, 2006. 28. 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TRANSFORMING GROWTH FACTOR-"1 PROTECTS AGAINST INTESTINAL EPITHELIAL BARRIER DYSFUNCTION CAUSED BY HYPOXIA-REOXYGENATION Kathryn L. Howe,*† Robert J. Lorentz,* Amit Assa,* Lee J. Pinnell,* Kathene C. Johnson-Henry,* and Philip M. Sherman*‡ *Cell Biology Program, Research Institute, Hospital for Sick Children, University of Toronto, Toronto; †Faculty of Health Sciences, Department of Surgery, Division of General Surgery, McMaster University, Hamilton, Ontario; and ‡Department of Paediatrics, Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada Received 3 Sep 2014; first review completed 22 Sep 2014; accepted in final form 5 Jan 2015 ABSTRACT—Intestinal epithelia regulate barrier integrity when challenged by inflammation, oxidative stress, and microbes. Transforming growth factor-"1 (TGF-"1) is a cytokine with known beneficial effects on intestinal epithelia, including barrier enhancement, after exposure to proinflammatory cytokines and infectious agents. The aim of this study was to determine whether TGF-"1 directly protects intestinal epithelia during hypoxia-reoxygenation (HR). Intestinal epithelial monolayers (T84, Caco-2bbe) were exposed to either hypoxia (1% O2, 1 h) or oxidative stress (hydrogen peroxide, 1 mM), followed by normoxic atmosphere for different time points in the absence and presence of varying concentrations of TGF-"1. Transepithelial electrical resistance (TER) assessed barrier function, with RNA extracted for reverse transcription polymerase chain reaction analysis of GPx-1, HIF-1, heme-oxygenase-1 (HO-1), and NOX-1. In some experiments, intestinal epithelia were exposed to enterohemorrhagic Escherichia coli (EHEC) O157:H7 during the reoxygenation period and TER recorded 7 h after the infectious challenge. Hypoxia-reoxygenation significantly decreased TER in intestinal epithelia compared with normoxic controls. Transforming growth factor-"1 pretreatment ameliorated HR-induced epithelial barrier dysfunction in T84 (at 1 Y 3 h) and Caco-2bbe (1 h) monolayers. Transforming growth factor-"1 preserved barrier integrity for up to 16 h after challenge with hydrogen peroxide. In TGF-"1Ytreated epithelial monolayers, only HO-1 mRNA significantly increased after HR (P G 0.05 vs. normoxic controls). The EHEC-induced epithelial barrier dysfunction was significantly worsened by intestinal HR (P G 0.05 vs. normoxia-EHECYinfected cells), but this was not protected by TGF-"1 pretreatment. Transforming growth factor-"1 preserves loss of epithelial barrier integrity caused by the stress of HR via a mechanism that may involve the upregulation of HO-1 transcription. Targeted treatment with TGF-" could lead to novel therapies in enteric diseases characterized by HR injury. KEYWORDS—Ischemia-reperfusion, intestinal permeability, cytokine, antioxidant
INTRODUCTION
enterohemorrhagic Escherichia coli (EHEC) O157:H7 (4). Transforming growth factor-" exerts protective effects in a murine model of colitis (5) and in several models of I/R injury, including myocardial infarction (6), stroke (7), and renal damage (8). Indirect evidence suggests that TGF-" also is protective in the setting of intestinal ischemia (9). Clinical studies demonstrate that TGF-" signaling is downregulated in IBD, a condition of inflammatory hypoxia, with SMAD7 antisense oligonucleotide treatment improving clinical signs and symptoms of active Crohn disease (10). Despite a common underlying mechanism for many of these conditions, it remains to be determined whether TGF-" directly protects intestinal epithelia in models of hypoxia and I/R. Given the properties of TGF-", we sought to determine using an epithelial cell culture model system whether 1) TGF- " protects against hypoxia-reoxygenation (HR)Yinduced intestinal epithelial barrier dysfunction, 2) HR exacerbates barrier dysfunction caused by a pathogenic bacterium, and 3) TGF-" protects against a double hit of exposure to both HR and an enteric pathogen.
Hypoxic conditions underlie the pathobiology of a variety of gastrointestinal conditions, including chronic inflammatory bowel diseases (IBDs), enteric infections, necrotizing enterocolitis, and ischemia-reperfusion (I/R) injury caused by mesenteric ischemia and organ transplantation (1). The epithelium lining the intestinal tract is composed of a highly metabolically active single layer of cells with both absorptive and barrier functions that exist in a state of physiologic hypoxia (2). The functional integrity of gut barrier function can be altered when challenged by proinflammatory cytokines, immune cells, oxidative stress, and pathogenic microbes. Transforming growth factor-" (TGF-") is an antiinflammatory cytokine with beneficial effects on intestinal epithelial barrier function, including barrier enhancement and protection against dysfunction caused by proinflammatory cytokines and infections, such as Cryptosporidium parvum (3) and Address reprint requests to Philip M. Sherman, MD, FRCPC, Peter Gilgan Centre for Research and Learning, 686 Bay St, Room 21-9430 Toronto, Ontario, Canada M5G 0A4. E-mail: [email protected]. This study was supported by the Canadian Institutes for Health Research (grants MOP-89894 and IOP-92890 to P.M.S.). P.M.S. is a recipient of the Canada Research Chair in Gastrointestinal Disease. None of the authors have any relevant conflicts of interest to report. DOI: 10.1097/SHK.0000000000000333 Copyright Ó 2015 by the Shock Society
METHODS Cell and bacterial culture T84 intestinal epithelial cells purchased from the American Type Culture Collection (Manassas, Va) were grown and maintained in a 1:1 (vol:vol) mixture of Dulbecco modified Eagle medium and Ham F-12 medium containing L-glutamine (0.365 g/L) and sodium bicarbonate (2.438 g/L), supplemented with 483
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FIG. 1. Transforming growth factor-"1, but not TGF- " 2, increases TER of polarized intestinal epithelia. Transforming growth factor-"1 (0.1 Y 100 ng/mL) added to the basolateral wells of either T84 (A) or Caco-2 (panel B) monolayers and TER was recorded after 24, 48, and 72 h. Values are expressed as percentage of control monolayers (A, n = 8 monolayers in four separate experiments [control TER 1,966 T 331, 2,145 T 435, and 1,899 T 490 4 & 0.33 cm2 for 24, 48, and 72 h, respectively). B: w/o denotes washout after 24 h, 10 ng/mL; n = 5 Y 11 monolayers in four experiments. #P G 0.05, *P G 0.01, **P G 0.001 vs. control monolayers (control TER 637 T 51, 548 T 51, and 520 T 53 4 & 0.33 cm2 for 24, 48, and 72 h, respectively). Transforming growth factor-"2 (10 ng/mL) was added to the basolateral well of T84 (C) or Caco-2 (D) monolayers, and TER was measured at 24-h intervals for up to 3 days. Values are expressed as percentage control monolayer TER. C, n = 9 monolayers in four experiments (control TER 2,263 T 609, 1,952 T 388, and 1,592 T 296 4 & 0.33 cm2 for 24, 48, and 72 h, respectively). D, n = 6 monolayers used in two experiments (control TER 859 T 50, 699 T 37, and 632 T 40 4 & 0.33 cm2 for 24, 48, and 72 h, respectively). P = N.S. versus control and between groups (ANOVA).
fetal bovine serum (10%) and 2% penicillin-streptomycin at 37-C in 5% CO2 (all from Invitrogen, Burlington, Ontario, Canada). Caco-2bbe (American Type Culture Collection, labeled Caco-2 hereafter) cells were grown and maintained in Dulbecco modified Eagle medium containing L-glutamine (0.584 g/L) and supplemented with 10% fetal bovine serum, 2% penicillin-streptomycin, sodium pyruvate (1 mM), and human transferrin (0.01 mg/mL) at 37-C in 5% CO2 (all from Invitrogen). Recombinant human TGF-"1 (TGF-" throughout text, unless otherwise specified), TGF-"2, or latent TGF-" (R&D Systems Inc, Minneapolis, Minn) were added into the basolateral side of wells (0.1 Y 100 ng/mL, 1 Y 72 h), and transepithelial electrical resistance (TER) was recorded throughout the experimental period. Enterohemorrhagic E. coli O157:H7 strain CL56 (11) was maintained on 5% sheep blood agar plates (Oxiod, Nepean, Ontario, Canada) at 4-C, cultured overnight in nonaerated Luria-Bertani broth at 37-C, and then added to the apical compartment of intestinal epithelia at approximately 1 108 colonyforming units/monolayer.
experiment, cells were placed in either warmed normoxic or hypoxic tissue culture medium for 60 min, TER was recorded (time 0), and reoxygenation was established by replacing each well with normoxic medium for up to 24 h. The TER was recorded at 1, 2, 3, and 24 h. In experiments testing the effect of TGF-", a single dose (10 ng/mL in normoxic culture medium) was added into the basolateral compartment 60 min before hypoxic challenge. For experiments determining whether HR exacerbates EHEC O157:H7Yinduced barrier dysfunction, epithelial monolayers were inoculated at the start of reoxygenation (time 0), and TER was recorded at 7 h after infection.
Oxidative stress T84 and Caco-2 cells were washed once in supplement-free culture medium and equilibrated for 1 h at 37-C in the absence or presence of TGF-" (10 ng/mL). Hydrogen peroxide (30% wt/wt; Sigma-Aldrich) was added to the apical surface of T84 or Caco-2 monolayers (50 2M Y 5 mM), and TER was recorded (multiple time points were measured up to 16 h).
Gene expression
Epithelial permeability Caco-2 and T84 cells were seeded onto the apical surface of semipermeable filter supports (surface area, 0.33 cm2 and 1 cm2, respectively; Corning Inc, Corning, NY) and cultured until the TER (measured with a Voltmeter and chopstick electrodes, Millicell-ERS; Millipore, Bedford, Mass) was greater than 1,000 4 cm2, which is indicative of tight junction formation. Changes in TER during the experimental protocol are indicative of alterations in epithelial barrier function. Paracellular permeability was also tested using a nonabsorbable fluoresceinisothiocyante (FITC)Ylabeled Dextran probe (10 kd; Sigma-Aldrich, St. Louis, Mo), as previously described (12). Briefly, FITC-Dextran (1 mg/mL in cell culture medium) was applied into the apical compartment of wells, and recovery of the probe in the basolateral side was measured after 1 h. Fluorescent units were calculated using a microplate fluorescence reader (Victor, Perkin-Elmer, Mass), and results were expressed as optical density arbitrary units.
Hypoxia-reoxygenation Hypoxic conditions were generated using a sealed modular chamber and flow meter system (Billups-Rothenberg Inc, Del Mar, Calif) flushed at 20 L/ min (5 min) with filtered 1% O2 (5% CO2, balance N2; Praxair, Danbury, Conn) (13). For hypoxic conditions, cell culture medium was placed into a vented T25 culture flask (Corning) and flushed with 1% O2, and the sealed chamber was placed in an incubator overnight at 37-C. At the start of an
Total RNA was extracted from cells grown on monolayers at the completion of experimental conditions using TRIzol Isolation Reagent, as per manufacturer’s instructions (Life Technologies, Burlington, Ontario, Canada). For HR experiments, samples were collected at 1 h after HR, whereas HRbacteria experiments were collected at 7 h after HR-EHEC challenge. Only samples with A260/280 more than 1.8 were used to synthesize cDNA using the iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, Calif). Primers were as follows: HIF-1!: forward, 5¶-TCACCACAGGACAG TACAGGATGC-3¶; reverse, 5¶-CCAGCAAAGTTAAAGCATCAGGTTCC-3¶ (14) GPx-1: forward, 5¶-CC AAGCTCATCACCTGGTCT-3¶; reverse, 5¶-TCGAT GTCAATGGTCTGGAA-3¶ (15) HO-1: forward, 5¶-AAGATTGCCCAGAAAGCCCTGGAC-3¶; reverse, 5¶-AACTGTCGCCACCAGAAAGCTGAG-3¶ (16) NOX-1: forward, 5¶-GGAGCAGGATTGGGGTCAC-3¶; reverse, 5¶TTGCTGTCCCATCCGGTGAG-3¶ (17) GAPDH: forward, 5¶-GTCATCCATGACAACTTTGG-3¶; reverse, 5¶-GAGCTTGACAAAGTGGTCGT-3¶ (14). Reverse transcription polymerase chain reaction was performed using SsoFast Eva-Green Supermix (Bio-Rad Laboratories) and CFX96 C1000 Thermal Cycler (Bio-Rad Laboratories). Values were normalized against the housekeeper gene GAPDH and then compared between experimental groups (18).
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FIG. 2. Hypoxia-reoxygenation decreases TER in intestinal monolayers. T84 (A) or Caco-2 (B) epithelial monolayers were exposed to either normoxia (open bars) or hypoxic conditions (1% O2, closed bar histograms) for 1 h, reoxygenated with normoxic tissue culture medium, and TER was recorded at 1, 2, 3, and 24 h. Values are expressed as TER (percentage of value at time 0) (A, n = 8 Y 15 monolayers used in three to six separate experiments; B, n = 8 Y 11 monolayers used in three to four experiments; #P G 0.05, *P G 0.01, **P G 0.001 vs. normoxic control [paired t test]). Note that, for ease of data legibility, the y axis originates at 80% of baseline TER.
Statistical analyses Data are presented as means T SEM. Data were analyzed using one-way analysis of variance (ANOVA), followed by post hoc comparisons using Tukey test, or using the paired Student t test, with P G 0.05 taken as the level of statistical significance.
RESULTS TGF-"1, but not TGF-"2, promotes epithelial barrier integrity
Using two intestinal epithelial cell lines, cryptlike T84 and villus-like Caco-2bbe, TGF-"1 (Fig. 1, A and B), but not TGF"2 (Fig. 1, C and D), enhanced barrier function in both a doseand a time-dependent manner. Accordingly, TGF-"1 was selected for use in all subsequent experiments (labeled hereafter as TGF-"). TGF-" preserves epithelial barrier dysfunction caused by HR
Using a modified HR model previously described by Xu et al. (13), HR significantly decreased intestinal epithelial barrier TER after 1, 2, 3, and 24 h of reoxygenation in T84 monolayers (Fig. 2A). As shown in Figure 3A, pretreatment with TGF-" or latent TGF-" ameliorated the effects of HR. Corroborating the findings of TER, HR also resulted in
increased paracellular permeability to a fluorescent-labeled macromolecular Dextran probe (Fig. 4). Using a complementary intestinal epithelium model, Caco2bbe (Caco-2) cells, we showed that the effects were not cell line specific. Hypoxia-reoxygenation also caused decreased TER of Caco-2 monlayers at 1 and 2 h after restoring normoxic conditions (Fig. 2B), an effect that was significantly ameliorated by TGF-" after 1 h of reoxygenation only (Fig. 3B). TGF-" ameliorates barrier dysfunction caused by H2O2
Hypoxia-reoxygenation produces downstream mediators including reactive oxygen species (ROS) known to cause barrier dysfunction (19, 20). Pretreatment with TGF-" for 1 h, but not the negative control, latent (inactive) TGF-", protected epithelial barrier dysfunction caused by H2O2 (1 mM) for up to 4 h in T84 (Fig. 5A) and at 3 and 16 h in Caco-2 monolayers (Fig. 5B). HR exacerbates epithelial barrier dysfunction caused by an enteric pathogen
In both T84 (Fig. 6A) and Caco-2 (Fig. 6B) monolayers, HR caused greater reductions in TER than observed with EHEC infection under conditions of normoxia.
FIG. 3. TGF-" improves HR-induced barrier dysfunction in intestinal epithelia. T84 (A) and Caco-2 (B) epithelial monolayers were treated with either TGF-"1 or latent TGF-" (10 ng/mL) for 1 h before HR challenge, and TER was then measured at 1, 2, 3, and 24 h. Values are expressed as the percentage of baseline TER at time 0. Normoxia alone is denoted in open bar histograms, normoxia plus TGF-"1 in crosshatch bar histograms, HR alone in closed bar histograms, HR + TGF-"1 in diamond bar histograms, and HR + latent TGF-" indicated by the horizontal line bar histograms (A, n = 6 Y 12 monolayers used in two to four experiments; B, n = 7 Y 9 monolayers tested in three separate experiments; #P G 0.05, *P G 0.01, **P G 0.001 vs. normoxic controls, lines above the bar histograms represent statistically significant differences between groups). For ease of data legibility, the y axis originates at 70% to 85% of baseline TER.
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FIG. 4. Hypoxia-reoxygenation increases paracellular permeability of polarized epithelia, which is ameliorated by TGF-". T84 monolayers were exposed to normoxia (left bar histogram) or hypoxia for 1 h (1% O2) either in the absence (middle bar histogram) or presence of TGF-"1 (10 ng/mL for 1 h) (right bar) followed by placing normoxic tissue culture medium containing FITCDextran (10 kd, 1 mg/mL) into the apical compartment. After 1 h, samples were collected from the basolateral compartment of polarized epithelia for optical density readings (n = 8 Y 10 monolayers in three to four experiments; *P G 0.01 vs. normoxia; line above the histograms represents a statistically significant difference between HR study groups).
TGF-"1 does not ameliorate the effect of HR on pathogen-induced barrier dysfunction
Pretreatment of T84 monolayers with TGF-" (10 ng/mL) for 1 h followed by HR and EHEC O157:H7 (7 h) did not prevent the drop in TER induced by the pathogen (54.6% T 4.9 % of baseline TER in the absence of TGF-" vs. 52.3% T 3.9% in the presence of TGF-"; n = 13 Y 14 monolayers tested in five separate experiments [data not shown]). TGF-" pretreatment increases expression of the antioxidant HO-1 in monolayers exposed to HR
To begin to elucidate mechanisms underlying TGF-" protection during HR, RNA was collected from intestinal epithelial monolayers. Levels of mRNA expression for GPx-1, NOX-1, and HIF1! were not significantly different between treatment groups (data not shown). By contrast, transcription of HO-1 was significantly altered by TGF-" treatment in gut monolayers exposed to HR (Fig. 7). DISCUSSION Intestinal ischemia and reperfusion injury is relevant to the pathophysiology of many gut diseases, with a common underlying
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process involving inflammatory mediators, epithelial barrier disruption, and bacterial translocation (21Y23). Proinflammatory cytokine mediators are upregulated in response to inflammatory or infectious insults in the intestine, which is balanced by concurrent upregulation of anti-inflammatory mediators that serve to quell the responses to injury. Transforming growth factor-" is a predominantly anti-inflammatory cytokine that regulates the function of many cells in the intestinal milieu, including epithelia, monocytes, and dendritic cells that interface with immune cells in the underlying lamina propria. Given the protective effect of TGF-" on intestinal epithelia (3, 4), we sought to determine if this antiinflammatory cytokine could ameliorate responses of intestinal epithelia to HR, an insult underlying many intestinal diseases. Studies using organ transplant, septic shock, and multiorgan failure models have advanced current understanding of mechanisms underlying I/R injury in the gut. Interest has more recently focused on the role of ongoing HR in chronic diseases, including IBDs (2). Importantly, the intestinal epithelium is uniquely able to withstand hypoxia through a mechanism involving upregulation of intestinal trefoil factor by HIF-1! (24). Models aimed at understanding these processes have begun to outline the inflammatory responses and barrier dysfunction caused not only by ischemia or hypoxia but also the ROS and intracellular signaling pathways generated in response (25). In this study, we continue to build on previous work by others (13, 15) showing that HR leads to epithelial barrier dysfunction after exposure of both cryptlike (T84) and villus-like (Caco-2bbe) intestinal cell lines and demonstrate that TGF-"1 pretreatment prevents HR-induced barrier dysfunction. Paracellular permeability studies corroborated these data, showing not only that HR increases permeability but also that TGF-" pretreatment for 1 h can prevent this effect. The protective effects of TGF-"1 on HR occurred at times that are well in advance of independent effects previously shown on barrier enhancement of the apical junction complexes, which are first observed after 16 h (4). Interestingly, the protective effect of a single hour-long pulse of TGF-" has been observed previously in a model of EHEC O157:H7Yinduced barrier dysfunction (4). In a murine model of chemical-induced colitis (Dextran sodium sulfate), recombinant TGF-" delivered to the gut protected against both acute and chronic injury (5), which likely reflects prevention of inflammation-induced HR. Importantly, phase I clinical trials have shown that restoration of
FIG. 5. TGF-" ameliorates disruption of TER caused by H2O2, an ROS produced by HR. T84 (A) and Caco-2 (B) monolayers were either left untreated (white bars) or pretreated with TGF-"1 (light gray bars) or latent TGF-" (1 h, 10 ng/mL, dark gray bars), equilibrated in plain medium (1 h), and subsequently treated with H2O2 (1 mM, apical well, black bars). Values are expressed as percentage of baseline TER (A, n = 3 Y 12 monolayers in up to three experiments; B, n = 4 monolayers used in two experiments; #P G 0.05, *P G 0.01 vs. normoxic controls, #*P G 0.01 vs. H2O2 and latent TGF-" + H2O2).
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FIG. 6. Hypoxia-reoxygenation exacerbates EHEC O157:H7Yinduced epithelial barrier dysfunction. T84 (A) and Caco-2 (B) polarized monolayers were exposed to hypoxic conditions (1% O2) for 1 h, reoxygenated with normoxic tissue culture medium in the absence or presence of EHEC O157:H7 (~1 108 colony-forming units/monolayer) placed into the apical compartment with TER measured 7 h after the infectious challenge. Values are expressed as percentage of TER measured at time 0 (A, n = 6 Y 15 monolayers used in three to five experiments, *P G 0.01, **P G 0.001, ***P G 0.0001, ANOVA, followed by paired Student t test versus normoxic controls; B, n = 6 monolayers in three experiments, **P G 0.01, paired Student t test).
TGF-" signaling in active Crohn disease is safe, well tolerated, and seems to improve disease activity (10). Our results add to this body of emerging data by showing that TGF-" is protective in gut HR. Reactive oxygen species are generated as a downstream event of HR and have direct damaging effects on the intestinal epithelium (26). Our study showed that pretreatment with TGF-" for 1 h prevented the barrier defect caused by direct H2O2 application to the T84 and Caco-2 cell lines. Although the preventive effect of TGF-" was statistically significant for up to 4 h in T84 monolayers, the continued trend persisted for several hours. In the Caco-2 monolayers, however, the effect of H2O2 was less robust and, notably, TGF-" was significantly protective at 3 and 16 h only. These data are compatible with those of Basuroy et al. (27) who demonstrated that epidermal growth factor prevents H2O2-induced barrier dysfunction in Caco-2 cells through direct action on the mitogen-activated protein kinase signaling pathway, ERK, and subsequent regulation of tight junction proteins and the actin cytoskeleton. The concentration of H2O2 (1 mM) we used is consistent with the optimal concentration determined by authors using the IPECJ2 small intestinal epithelial cell line (28). Interestingly, in contrast to findings using H2O2, latent TGF-" protected against HR-induced barrier dysfunction, despite its planned use as a negative control. Although activators of latent TGF-" in vitro are extreme pH, heat, and ionizing radiation, none of these were factors during the 60-min application of the latent cytokine. One possible explanation for the discrepancy is that, despite washout, residual latent TGF-" was activated during HR exposure, thereby releasing active TGF-" from the latency-associated peptide. Unlike the global effects of HR on cell monolayers, H2O2 was applied directly to the apical surface where contact with basolaterally applied latent TGF-" was physically excluded. Consequently, the inactive form of TGF-" was not protective against H2O2-induced epithelial barrier dysfunction. The HR-induced barrier dysfunction model was also used to test the effects of additional insults by exposing cell lines to EHEC O157:H7. Hypoxia-reoxygenation exacerbated the barrier defects caused by exposure to the microbe alone, findings consistent with a previous study demonstrating exposure to hypoxia and nonpathogenic E. coli followed by 3 h of normoxia caused permanent barrier disruption in rat mucosal ileal sections,
unlike the reversible effect caused by hypoxia alone (29). Although they used a putative nonpathogenic strain, we chose to test enteropathogenic bacteria because these predominate in settings of sepsis and other chronic inflammatory conditions where the diversity of the normal gut microbiota has been reduced by the use of antibiotics (30). Epithelia placed under pharmacologically induced metabolic stress also have compromised barrier function when exposed to commensal bacteria (31), supporting the hypothesis that stress exacerbates host-microbial interactions. Given the role of TGF-" in preserving epithelial barrier during I/R injury (9) and protecting against colitis in mice with genetically engineered barrier compromise (32), we hypothesized that pretreatment with TGF-" would protect against the double insult of HRYpathogenic bacteria. However, the data were not supportiveVsurprising given that TGF-" ameliorates epithelial barrier disruption caused by HR and the ROS product, H2O2, and that it prevents barrier defects caused by EHEC O157:H7 (4). It is possible that the virulence of pathogenic bacteria in the setting of HR may be too great an insult to overcome with TGF-" pretreatment. Indeed, recent studies have determined that HR causes intestinal epithelial release of adenosine and dynorphin and direct activation of the quorum sensing circuitry of Pseudomonas aeruginosa, leading to increased expression of virulence determinants and greater barrier dysfunction (33). This work has since been corroborated
FIG. 7. TGF-" increases epithelial HO-1 gene expression in the setting of HR. T84 monolayers were pretreated with TGF-"1 (10 ng/mL for 1 h) before challenge with HR. RNA was collected after 1 h of reoxygenation, and HO-1 mRNA expression was measured relative to the housekeeping gene GAPDH. n = 8 Y 12 monolayers tested in three separate experiments. Horizontal line above the bar histograms represents a difference between groups, *P G 0.05 by ANOVA.
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in vivo, where I/R causes 100% mortality in mice cochallenged with P. aeruginosa compared with less than 10% for bacterial challenge in the absence of I/R or less than 50% for I/R alone (34). In contrast to the HR-pathogen experiments, it is noteworthy that, despite the greater barrier insult caused by H2O2, TGF-" was protective for a period, suggesting the highly relevant contribution of microbes to the regulation of gut permeability. We show that HO-1 mRNA is upregulated at a time when TGF-" protective effects during HR are evident. This is consistent with previous reports indicating that HO-1 is a mediator of gastroprotective pathways (35) and that increased HO-1 expression protects against I/R injury in the intestine (36). More recently, in a mouse model of necrotizing enterocolitis, HO-1 deficiency was shown to promote the development of intestinal injury after oral challenge with formula and exposure to HR (37). Previous studies show that both curcumin and hirsutenone ameliorate oxidant-induced intestinal epithelial barrier disruption by upregulating HO-1 (20). Future studies, therefore, should determine whether limiting induction of HO-1(using either a chemical inhibitor or silencing RNA) blocks the protective effects of TGF-" in the setting of HR. Taken together, the findings in this report show that TGF-" protects against HR-induced intestinal barrier dysfunction, as well as that induced by the ROS H2O2. A mechanism for this beneficial effect may involve the upregulation of HO-1 specifically. By contrast, TGF-" was not able to protect against the exacerbated barrier dysfunction caused by HR and enteric bacterial pathogens. These studies emphasize the diverse impacts of HR on intestinal epithelia and propose a means that could be used, at least in some clinical settings, to ameliorate such effects. Targeted TGF-" therapy holds promise for use in managing intestinal disease processes characterized by HR.
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