1 University of Calgary and 2 Surgical Medical Research Institute, 3 Division of Gastroenterology, and 4 Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta T6G 2C2, Canada
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ABSTRACT |
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Crohn's disease is a chronic disease
characterized by oxidant-induced tissue injury and increased intestinal
permeability. A consequence of oxidative damage is the accumulation of
DNA strand breaks and activation of poly(ADP-ribose) polymerase (PARP),
which subsequently catalyzes ADP-ribosylation of target proteins. In this study, we assessed the role of PARP in the colitis seen in interleukin (IL)-10 gene-deficient mice. IL-10 gene-deficient mice
demonstrated significant alterations in colonic cellular energy status
in conjunction with increased permeability, proinflammatory cytokine
release, and nitrosative stress. After 14 days of treatment with the
PARP inhibitor 3-aminobenzamide, IL-10 gene-deficient mice demonstrated
normalized colonic permeability; reduced tumor necrosis factor- and
interferon-
secretion, inducible nitric oxide synthase
expression, and nitrotyrosine levels; and significantly attenuated
inflammation. Time course studies demonstrated that 3-aminobenzamide
rapidly altered cellular metabolic activity and decreased cellular
lactate levels. This was associated with normalization of colonic
permeability and followed by a downregulation of proinflammatory cytokine release. Our data demonstrate that inhibition of PARP activity
results in a marked improvement of colonic inflammatory disease and a
normalization of cellular metabolic function and intestinal permeability.
interleukin-10; 3-aminobenzamide; inflammatory bowel disease; intestinal permeability
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INTRODUCTION |
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UNDER NORMAL PHYSIOLOGICAL conditions, the intestinal epithelium acts as a selective barrier, allowing uptake of nutrients and water but limiting the passage of luminal antigens into the lamina propria. The barrier function of the intestine is regulated at the level of the tight junctions close to the apical surface of epithelial cells (30). Tight junctions are dynamic, energy-requiring protein structures that can be modulated by bacterial products (12, 14, 15), dietary constituents (2, 23), immunological factors (26, 36), alterations in cellular energy status (29, 30, 44), and pH (31, 39). Disruption of tight junctions leads to increases in epithelial permeability and abrogated barrier function.
The lumen of the intestine contains bacteria, bacterial products, and dietary antigens capable of initiating and sustaining inflammation. Disruption of the epithelial barrier, particularly in the crypt region, could lead to aberrant antigen uptake, an uncontrolled activation of the mucosal immune system, and subsequent intestinal inflammation (11). Patients with Crohn's disease have increased intestinal permeability, either as a primary defect or as an acquired defect secondary to intestinal inflammation (8, 16, 35). This defect in intestinal barrier function has been postulated to play a role in the exacerbation of intestinal inflammation in Crohn's disease by allowing increased antigen uptake and a continuous stimulation of the mucosal immune system.
The disruption in barrier function of the intestine during active
inflammation has been linked to the generation of reactive oxygen and
nitrogen species and resultant tissue injury (13). High
levels of superoxide (SO) and nitric oxide (NO) have been described in
areas of active inflammation (13). These can either directly effect tissue injury or react together to produce the potent
oxidant peroxynitrite (ONOO) (4).
Peroxynitrite can cause oxidative damage and DNA strand breakage within
epithelial cells (41). One consequence of oxidative damage
is the activation of the nuclear enzyme poly(ADP-ribose) polymerase
(PARP; EC 2.4.2.30) (22). This enzyme acts to transfer the
ADP-ribose moiety of NAD+ to various chromatin proteins
(43). Chronic activation of PARP can potentially lead to a
depletion of cellular NAD+ and, because NAD+ is
necessary for ATP synthesis, a decline in cellular ATP levels and
potential cell dysfunction (43). Indeed, in vitro exposure of intestinal epithelial monolayers to a peroxynitrite donor results in
PARP activation, ATP depletion, and decreased transepithelial resistance (19).
Currently, strategies for treatment of inflammatory bowel disease
remain focused on suppression of the cellular inflammatory response.
However, these approaches to anti-inflammatory therapy, including the
use of monoclonal antibodies against tumor necrosis factor (TNF)- or
the use of the anti-inflammatory cytokine interleukin (IL)-10, remain
suboptimal. Because the disrupted mucosal barrier function seen in
patients with inflammatory bowel disease almost certainly acts to
exacerbate intestinal inflammation by allowing increased antigenic
uptake, we predict that therapy aimed at correcting such a permeability
defect would have a beneficial effect in treating chronic inflammation.
In the present study, we have assessed the role of PARP in the increased permeability that is associated with chronic, nonresolving colitis that develops spontaneously in the IL-10 gene-deficient mouse (5, 28). Here we demonstrate that pharmacological inhibition of PARP activity results in a rapid normalization of cellular metabolic function and intestinal permeability followed by a dramatic improvement in colonic inflammatory disease.
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MATERIALS AND METHODS |
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Materials
Chemicals of reagent grade were obtained from Sigma Chemical (St. Louis, MO) or Fisher Scientific (Napean, ON, Canada). The radioisotopes [14C]polyethylene glycol and D-[3H]mannitol were obtained from New England Nuclear (Boston, MA). ELISA kits used for the measurement of TNF-Animals
Homozygous IL-10 gene-deficient mice, generated on a 129 Sv/Ev background, and 129 Sv/Ev controls were housed under specific pathogen-free conditions. All provisions for the facility were autoclaved. Nonautoclavable supplies were sprayed with disinfectant and introduced through a HEPA-filtered air lock. Mice were housed in microisolator cages with tight-fitting lids containing a spun polyester fiber filter. In sentinel BALB/c mice, bacterial cultures, parasitological examinations, serological tracking profiles, and histological stains were negative for known murine viral and bacterial pathogens, indicating that the barrier was intact.Experimental Groups
Chronic.
IL-10 gene-deficient mice were treated with the PARP inhibitor
3-aminobenzamide (20 mg/kg) via an intraperitoneal injection for 14 days, starting at 8 wk of age (a time when colitis is firmly established). A vehicle-treated group received 3-aminobenzoic acid, an
inactive structural analog of 3-aminobenzamide with no inhibitory
effect on PARP activity (3). Food intake was monitored, and weight gain was recorded over the 14-day treatment period. Colons
from mice were assessed histologically, and colonic permeability, TNF- and IFN-
secretion, and nitrotyrosine, ATP, ADP, AMP, and lactate levels were measured. In addition, wet weights of heart, lungs,
liver, kidneys, and small and large intestine were recorded, as were
small and large intestinal lengths.
Acute.
Three-week-old IL-10 gene-deficient mice do not demonstrate any
histological signs of inflammation or any neutrophilic influx into the
lamina propria but do show increased colonic permeability (28). To determine the acute effects of 3-aminobenzamide
treatment in the absence of significant ulceration, IL-10
gene-deficient mice were injected with either saline (0.2 ml/kg) or
3-aminobenzamide (20 mg/kg i.p. in 0.2 ml/kg saline). At 2 and 4 h
following injection, separate groups of mice were anesthetized for
measurement of colonic permeability, TNF- and IFN-
secretion, or
cellular ATP and lactate levels.
Intestinal Histological Assessment
Animals were killed using pentobarbital sodium (160 mg/kg). Segments of colon were harvested, fixed in 10% phosphate-buffered formalin, paraffin embedded, sectioned at 4 µm, and stained with hematoxylin and eosin for light microscopic examination. The slides were reviewed independently by two pathologists in a blinded fashion (A. Wessler and L. D. Jewell) and assigned a histological score for intestinal inflammation using a scheme adapted from Saverymuttu et al. (40) and detailed in Table 1. Histological grades represent the numerical sum of four scoring criteria: mucosal ulceration, epithelial hyperplasia, lamina propria mononuclear infiltration, and lamina propria neutrophilic infiltration.
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Intestinal Permeability Measurements
In vivo perfusion. On the day of study, mice were premedicated with atropine (0.2 mg/kg) followed by an intraperitoneal injection of Hypnorm (25 mg/kg) and midazolam (12.5 mg/kg). In vivo absorption was measured by a single-pass perfusion technique, as previously described (32). In brief, the entire colon was isolated and cannulated at the proximal and distal ends. The gut was flushed with isosmotic Tyrode buffer (in g/l: 8 NaCl, 0.2 KCl, and 0.33 NaH2PO4, pH 7.4) to clear luminal contents. The segment was then perfused with test solution containing 5 g/l polyethylene glycol 4000 and 1 mM D-mannitol prepared in Tyrode buffer and radiolabeled with [14C]polyethylene glycol (10 µCi/l) and D-[3H]mannitol (100 µCi/l). Preliminary experiments determined that polyethylene glycol 4000 was not absorbed in either control or IL-10 gene-deficient mice (data not shown). The solution was perfused at a constant rate of 0.2 ml/min, and body temperature was maintained at 37°C. Intraluminal hydrostatic pressure was constantly monitored. After a 30-min equilibration period, six consecutive 10-min perfusion samples were collected from the distal site. The samples were weighed, and aliquots were taken for liquid scintillation counting. After completion of the procedure, animals were killed and the perfused segment of intestine was removed; its length was measured, and then it was dried for determination of dry weight.
Intestinal permeability.
Net water flux was calculated on the basis of differences between
initial and final volumes of perfusate and by the differences between
initial and final concentrations of [14C]polyethylene
glycol according to the following equation
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Mucosal Cytokine Secretion
Colonic organ cultures were prepared from control mice, IL-10 gene-deficient mice, and IL-10 gene-deficient mice treated with 3-aminobenzamide (20 mg · kgNO Synthase Activity
Colonic mucosa was homogenized on ice in a buffer composed of (in mM) 50 Tris, 0.1 EGTA, 0.1 EDTA, 12 2-mercaptoethanol, and 1 phenylmethylsulfonyl fluoride (pH 7.4). The homogenate was incubated with a cation-exchange resin (AG 50W-X8, Na+ form) for 5 min at 4°C to deplete endogenous L-arginine. Conversion of L-[3H]arginine to L-[3H]citrulline was measured in homogenates. Experiments in the presence of NADPH, without calcium and with 5 mM EGTA, were performed to determine the calcium-independent NO synthase (NOS) activity. Protein concentration was determined by the Bradford method (6).Western Blotting
Colonic mucosa was suspended in 0.5 ml Mono Q buffer (50 mMMetabolite Assays
Whole colons were snap frozen using Wollenberg clamps precooled in liquid nitrogen and subsequently stored atMeasurement of PARP Activity
Colons were removed, and the mucosa was scraped. The resultant tissue pellet was analyzed for PARP activity using a commercial activity assay (Genzyme Diagnostics, Cambridge, MA). Cell suspensions from mucosa were prepared by gentle physical disruption in ice-cold buffer (in mM: 50 Tris · HCl, pH 8, 25 MgCl2, and 0.1 phenylmethylsulfonyl fluoride), followed by centrifugation at 3,000 g for 5 min at 4°C. Twenty micrograms of total protein were transferred from the supernatant to microcentrifuge tubes at room temperature. The reaction was started with the addition of 1 mM NAD and 2 µCi [32P]NAD. The reaction was allowed to proceed for 1 min and then stopped by the addition of 900 µl of 20% ice-cold TCA. The contents of the reaction tube were filtered through a prewetted glass fiber filter under vacuum and washed four times with 3 ml of 10% ice-cold TCA and twice with 3 ml of 95% cold ethanol. The filters were then dried and counted. PARP activity is expressed as nanomoles per minute per microliter.Statistical Analysis
Data are reported as means ± SE. Differences between mean values were evaluated using analysis of variance or paired t-tests where appropriate (SigmaStat; Jandel, San Rafael, CA). Specific differences were evaluated using the Student-Newman-Keuls test. ![]() |
RESULTS |
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Chronic Treatment of IL-10 Gene-Deficient Mice
Animal profile and histological assessment.
Over the 14-day treatment period, control mice and IL-10 gene-deficient
mice receiving 3-aminobenzamide gained weight (Table 2). In contrast, despite having a similar
food intake, IL-10 gene-deficient mice lost weight, resulting in a
significantly reduced total body weight compared with controls. On the
other hand, small intestinal length and weight were increased in IL-10 gene-deficient mice compared with controls, and these were reduced by
3-aminobenzamide treatment. There was no difference in large intestinal
length among the three groups; however, large intestinal weight was
significantly increased in IL-10 gene-deficient mice. Again,
3-aminobenzamide treatment reduced colonic weight in the IL-10
gene-deficient mice. Wet weights of heart, lung, liver, and kidney did
not differ between groups (data not shown).
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Cytokine measurement.
Previous studies have shown that immune cells found in colonic tissue
from IL-10 gene-deficient mice spontaneously produce higher levels of
proinflammatory cytokines compared with immune cells from wild-type
mice (5). To determine whether the improvement in
histological score induced by 3-aminobenzamide treatment was accompanied by an alteration in cytokine production, TNF- and IFN-
secretion were measured. As previously shown (28),
colons from IL-10 gene-deficient mice spontaneously produced higher
levels of TNF-
and IFN-
compared with control mice (Fig.
2), likely because of the large influx of
lymphocytes into the lamina propria. In addition, IL-10 gene-deficient
mice also produced higher levels of TNF-
and IFN-
in response to
LPS stimulation compared with control mice. In correlation with the
attenuation of inflammation and reduction of lymphocytic infiltration,
spontaneous and LPS-stimulated colonic secretion of both TNF-
and
IFN-
were significantly reduced in those mice receiving
3-aminobenzamide for 2 wk (Fig. 2). Furthermore, in control and
3-aminobenzamide-treated mice, there was no significant difference in
spontaneous and LPS-stimulated cytokine release. In contrast, in IL-10
gene-deficient mice, LPS-stimulation resulted in a significant increase
in proinflammatory cytokine release, suggesting the presence of
monocytes within the lamina propria (Fig. 2).
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NOS assessment.
NO is produced by two distinct isoforms in epithelial tissue.
Constitutive NOS (cNOS) is a calcium-dependent constitutive enzyme,
whereas iNOS is a calcium-independent isoform whose expression is
induced in response to various insults, including TNF-
(34). iNOS activity was assessed enzymatically, and
protein levels were assessed by Western blotting. Colonic tissue from
IL-10 gene-deficient mice demonstrated high levels of iNOS activity
(Fig. 3A) and iNOS protein
expression (Fig. 3B). Both iNOS activity and protein levels were significantly reduced after 2 wk of 3-aminobenzamide treatment. This reduction of iNOS activity likely reflects a decrease in iNOS-expressing cells in the lamina propria, since histological assessment revealed a significant decrease in lymphocytic infiltration after 3-aminobenzamide treatment. On the other hand, cNOS activity was
not different among the three groups (data not shown).
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Nitrotyrosine formation.
The interaction of proinflammatory cytokines with specific receptors on
the plasma membranes of phagocytes can lead to the activation of the
plasma membrane-associated flavoprotein NADPH oxidase and the
subsequent release of large amounts of SO and hydrogen peroxide. SO can
then react with NO to form the very potent oxidant peroxynitrite
(18). IL-10 gene-deficient mice demonstrated increased
levels of TNF- and IFN-
and iNOS activity. This, coupled with the
very large influx of leukocytes into the lamina propria (see Table 3),
could result in high concentrations of reactive oxygen and nitrogen
species (9). The formation of nitrotyrosine adducts in
tissue serves as an indicator of the presence of reactive nitrogen
species (17). Assessment of total levels of nitrotyrosine
by Western blotting revealed evidence of increased levels of
nitrosylated proteins in IL-10 gene-deficient mice, as exemplified by
the appearance of a new nitrosylated protein at ~70 kDa and an
increase in the levels of a protein migrating at ~25 kDa (Fig.
4). In conjunction with the reduced
levels of TNF-
and iNOS activity, 3-aminobenzamide-treated IL-10
gene-deficient mice also had significantly reduced levels of the 70-kDa
nitrotyrosine band. The identity of these proteins is unknown, although
previous studies have also described a low-molecular-weight
nitrosylated protein that migrates close to 25 kDa (7).
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PARP activity.
Reactive oxygen and nitrogen species are able to indiscriminately
attack DNA, resulting in DNA strand breakage (47). This DNA damage subsequently activates the nuclear enzyme PARP
(43). Thus, to determine whether the presence of increased
levels of nitrotyrosine in IL-10 gene-deficient mice was associated
with enhanced levels of PARP activity, mucosa was scraped and PARP activity was measured. As seen in Fig. 5,
PARP activity was enhanced approximately fourfold in 10-wk-old IL-10
gene-deficient mice compared with controls. 3-Aminobenzamide treatment
for 14 days completely normalized PARP activity in IL-10 gene-deficient
mice (Fig. 5).
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Colonic permeability.
In vitro studies have demonstrated that increased paracellular
permeability can occur as a result of exposure to exogenous NO or
peroxynitrite (19, 43). Moreover, inhibition of PARP activity prevents the increase in paracellular permeability induced by
peroxynitrite (19). To determine whether inhibition of
PARP activity in vivo would have a similar effect in a chronic model of
colitis, colonic permeability was measured in mice using a single-pass
perfusion technique. At 10 wk of age, IL-10 gene-deficient mice had
significantly increased colonic permeability and decreased water
absorption compared with age-matched control mice (Fig. 6). On the other hand, IL-10
gene-deficient mice treated with 3-aminobenzamide for 14 days displayed
normal colonic permeability and water absorption (Fig. 6).
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Metabolite levels.
Levels of ATP, ADP, AMP, and lactate were measured in whole colon
homogenates to determine whether the increase in colonic permeability
observed in IL-10 gene-deficient mice was associated with altered
energy status. Compared with control mice, IL-10 gene-deficient mice
had significant alterations in colonic cellular energy status,
demonstrating increases in cellular ADP and lactate (P < 0.05) and suggesting an apparent shunting of energy production from
oxidative phosphorylation to anaerobic glycolysis (Table 4). Interestingly, total levels of cellular ATP were
increased in colons from IL-10 gene-deficient mice compared with
control colons. Inhibition of PARP activity with 3-aminobenzamide
resulted in decreases in cellular ADP and lactate and a further
increase in cellular ATP levels and cellular energy charge (Table 4). Thus disease in IL-10 gene-deficient mice was not associated with decreased total energy charge but was associated with high levels of
lactate. This would suggest that lactate acidosis and not reduced levels of ATP may be involved in alterations of intestinal
permeability.
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Acute Treatment of IL-10 Gene-Deficient Mice
There is evidence that inhibition of PARP reduces the recruitment of neutrophils into intestinal tissue by inhibiting the expression of adhesion molecules (48-50). In addition, inhibition of PARP activity can limit the release of proinflammatory cytokines from macrophages (21). Thus the improvement of inflammation in IL-10 gene-deficient mice treated for 14 days may have been initiated either by a 3-aminobenzamide-induced inhibition of neutrophil infiltration into the lamina propria or a 3-aminobenzamide-induced downregulation of cytokine release. Alternatively, if epithelial permeability was corrected first by a 3-aminobenzamide effect on cellular energetic status, this would reduce the level of antigenic materials entering the lamina propria and thus remove the antigenic trigger that may stimulate neutrophilic influx and cytokine release.We examined the effects of a single injection of 3-aminobenzamide in
3-wk-old IL-10 gene-deficient mice to determine a time course of
3-aminobenzamide-induced changes in cellular energy status,
permeability, and proinflammatory cytokine release. At 3 wk of age,
IL-10 gene-deficient mice exhibit an increase in colonic permeability,
with no histological evidence of neutrophilic infiltration or
ulceration (28). However, mice at this age do demonstrate
increased TNF- and IFN-
secretion. As seen in Fig. 7C, a single injection of
3-aminobenzamide produced a significant reduction in colonic mannitol
clearance in 3-wk-old IL-10 gene-deficient mice by 2 h. Coinciding
with the improvement in permeability were decreases in cellular lactate
levels (Table 5). Total cellular ATP
levels were not different among controls, IL-10 gene-deficient mice,
and IL-10 gene-deficient mice receiving 3-aminobenzamide either at 2 or
4 h after injection. In contrast to the rapid effects of
3-aminobenzamide treatment on cellular metabolic function and permeability, TNF-
(Fig. 7A) and IFN-
(Fig.
7B) secretion were not significantly reduced until 4 h
after 3-aminobenzamide injection. This would suggest that inhibition of
PARP activity with 3-aminobenzamide has a direct effect on cellular
metabolic function independent of and before a suppression of
proinflammatory cytokine release and neutrophilic influx.
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DISCUSSION |
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In this study, we demonstrate for the first time that established colonic inflammatory disease can be effectively treated by pharmacological inhibition of PARP activity. In addition, we have demonstrated a new mechanism for the beneficial effects of PARP inhibition, that being a normalization of cellular metabolic function and intestinal permeability.
Colitis in the IL-10 gene-deficient mouse develops shortly after
weaning at ~4 wk of age (27). By 8 wk of age, colitis is well established and is characterized by patchy mucosal ulceration, extensive neutrophilic infiltration into the lamina propria, and epithelial hyperplasia (5). Disease in IL-10
gene-deficient mice is initiated by CD4+ T cells and
results in an IL-12- and IFN--directed excessive generation and
activation of Th1 cells (5). Similar to what is seen in
patients with Crohn's disease, colonic inflammation in this model is
associated with increased permeability, high levels of mucosal IFN-
and TNF-
, and increased NO production (28). The cause
of the increased permeability in IL-10 gene-deficient mice exhibiting
colonic inflammation may be due to increased levels of peroxynitrite, a
reaction product of NO and the SO anion and a common effector of tissue
injury during inflammation (4, 47). In this study, we
found evidence for elevated levels of mucosal nitrotyrosine in colonic
tissue from adult IL-10 gene-deficient mice, which is suggestive of
nitrosative stress and a reaction between peroxynitrite and tyrosine
residues (4). Indeed, NO has been shown in vitro to
mediate IFN-
-induced increases in permeability (45),
and, furthermore, Kennedy et al. (19) have reported that
nitrosative stress increases epithelial permeability in intestinal
monolayers via DNA damage and a subsequent activation of the enzyme
PARP. In conjunction with the increased levels of nitrotyrosine, we
also observed an elevation of colonic PARP activity.
The mechanism behind a PARP-induced increase in epithelial permeability has been suggested to be a reduction in cellular ATP levels and a resultant breakdown of tight junctional integrity (43). Indeed, this has clearly been shown to occur in intestinal monolayers in vitro through a PARP-induced depletion of cellular NAD+ and a subsequent suppression of ATP synthesis (19). However, we were unable to demonstrate a similar reduction in tissue ATP levels in vivo. In fact, colonic homogenates from IL-10 gene-deficient mice demonstrated higher levels of cellular ATP compared with control mice. This may be due to the fact that we examined whole tissue ATP levels, which reflect ATP levels of numerous cell types. In addition, diseased mice have a high number of recruited immune cells present in the lamina propria. However, we did observe significant alterations in ADP and lactate levels in colonic tissue from both 3-wk-old and 10-wk-old IL-10 gene-deficient mice, suggesting an increased demand for ATP and a shunting of energy production from oxidative phosphorylation to anaerobic glycolysis both before the development of histological inflammation (3 wk old) and in the presence of active inflammation (10 wk old). Although the reason for this shunting is unknown, it has previously been shown that an active inflammatory state results in elevated energy expenditure and enhanced protein catabolism (20, 24). Indeed, IL-10 gene-deficient mice exhibited a net weight loss over the 14-day treatment period, despite consuming the same amount of food as did control mice. The length of the small intestine was also significantly increased in IL-10 gene-deficient mice, likely as an adaptation to this hypermetabolic state. Oxidative damage to mitochondria could also cause shunting of energy production toward anaerobic glycolysis. IL-10 gene-deficient mice exhibit high levels of proinflammatory cytokines and nitrotyrosine; thus a loss of function of mitochondria in these mice is very possible. Furthermore, activation of PARP itself can inhibit mitochondrial activity. Indeed, we found that inhibition of PARP rapidly restored lactate levels and colonic permeability to normal in both 3-wk-old and 10-wk-old mice, suggesting the involvement of PARP activity. Activated PARP consumes the ADP ribose moiety of NAD+ to synthesize ADP-ribose polymers on a variety of nuclear target proteins, including DNA polymerase I, histones, and PARP itself in a futile cycle that can deplete cellular NAD+ (22). This PARP-induced depletion of cellular NAD+ can inhibit mitochondrial respiration and result in enhanced levels of anaerobic glycolysis (22). Interestingly, the increased levels of cellular lactate suggest the presence of cellular acidosis, and it may be the acidosis that is altering intestinal permeability in vivo, as has been seen in endotoxin-induced mucosal acidosis in pigs (39). Indeed, low extracellular pH has been shown to increase epithelial permeability in Caco-2BBe cells (31) and potentiate NO-induced increases in intestinal permeability in vitro (44). Further studies are necessary to determine exactly which cells (epithelia, fibroblasts, immune) are exhibiting alterations in metabolic function in the colons of IL-10 mice and the role of PARP activity in these alterations.
Previous studies have clearly shown that pharmacological inhibition of PARP activity is beneficial in various acute models of inflammation, such as stroke (43), endotoxic shock (42), and ischemia-reperfusion injury (49). In addition, Zingarelli and co-workers (50) demonstrated that PARP-deficient animals are protected from the lethal effects of trinitrobenzene sulfonic acid (TNBS) colitis. However, unlike the IL-10 gene-deficient mouse model, TNBS colitis is not spontaneous and is ultimately resolved in time (33). In contrast, in this study we demonstrate that PARP inhibition is beneficial in a chronic model of colitis and that treatment of established colitis by pharmacological inhibition of PARP dramatically improves barrier and metabolic function and, subsequently, inflammation.
There are several mechanisms by which long-term inhibition of PARP activity may lead to resolution of colitis. Zingarelli et al. (49, 50) suggested that inhibition of PARP activation reduces intracellular adhesion molecule-1 and P-selectin expression, leading to an inhibition of neutrophil recruitment and decreased oxidant generation in the lamina propria. Our data showing that neutrophil infiltration and nitrotyrosine formation are significantly reduced after 14 days of treatment with 3-aminobenzamide support this hypothesis. However, in our study we show that intestinal permeability is enhanced in 3-wk-old mice in the absence of any neutrophilic infiltration. Furthermore, a single injection of 3-aminobenzamide can restore metabolic alterations and intestinal permeability to normal within 2 h, arguing against the notion that the primary beneficial effects of PARP inhibition are due to changes in adhesion molecule expression.
In addition to NO and peroxynitrite, proinflammatory cytokines such as
IFN- and TNF-
also increase permeability in epithelial monolayers, albeit possibly through a NO-mediated mechanism (26, 36). Colonic secretion of both IFN-
and TNF-
are increased in IL-10 gene-deficient mice before the development of histological inflammation and neutrophilic infiltration (28),
suggesting the presence of activated T cells and macrophages. In
macrophages, an ADP-ribosylation reaction mediated by PARP appears to
contribute to the activation of nuclear factor (NF)-
B and precedes
the upregulation of iNOS activity and release of proinflammatory
cytokines (20). Inhibition of PARP prevents the release of
these cytokines and, indeed, attenuates NF-
B activation in
macrophages (21). Thus treatment of mice with
3-aminobenzamide in vivo may downregulate cytokine release from
macrophages and, in this context, improve permeability. However, the
acute studies clearly demonstrate that 2 h after a single
injection of 3-aminobenzamide, colonic permeability is significantly
reduced, whereas attenuation of TNF-
and IFN-
secretion did not
occur until 4 h after 3-aminobenzamide injection. Nonetheless, the
inhibition of proinflammatory cytokine release from macrophages likely
contributes to the beneficial effects of PARP inhibition.
Another potential mechanism by which PARP inhibition may act to improve barrier function and suppress inflammation is the prevention of necrosis by colonic epithelial cells following oxidant-mediated injury (46). It has been suggested that a lack of sufficient ATP results in a damaged cell undergoing necrosis, a proinflammatory event, rather than apoptosis (46). However, our acute studies demonstrating that PARP inhibition attenuates the permeability defect within 2 h after injection would suggest that prevention of necrosis is not the initiating factor in the suppression of inflammation, but, again, this may contribute to the beneficial effects of PARP inhibition.
Together, these data support a role for PARP in the alteration of cellular metabolic function and intestinal permeability in the IL-10 gene-deficient mouse. Although it is difficult to determine from this study the relative contribution of various PARP activation pathways (i.e., enterocyte, endothelial cell, immune cell) to mucosal inflammation, this work does highlight a role for PARP and poly ADP-ribosylation mechanisms in the perpetuation of chronic inflammation. Indeed, this study demonstrates that inhibition of PARP results in a dramatic improvement of mucosal function in a chronic model of intestinal inflammation. This likely occurs through multiple mechanisms, including a rapid restoration of epithelial barrier integrity through a correction of cellular metabolic function. Therefore, this study supports PARP inhibition as a therapeutic tool in the treatment of inflammatory bowel disease.
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ACKNOWLEDGEMENTS |
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This work was supported by the Alberta Heritage Foundation for Medical Research, the Crohn's and Colitis Foundation of Canada, and Schering Canada, which supported the IL-10 gene-deficient mouse colony.
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FOOTNOTES |
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Address for reprint requests and other correspondence: K. L. Madsen, Univ. of Alberta, 536 Newton Bldg., Edmonton, Alberta T6G 2C2, Canada (E-mail: karen.madsen{at}ualberta.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 17 April 2000; accepted in final form 13 June 2000.
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