Intestinal alkaline phosphatase (IAP) is a small intestinal brush border enzyme that has been shown to function as a gut mucosal defense factor, but its precise mechanism of action remains unclear. We investigated the effects of IAP on specific bacteria and bacterial components to determine its molecular targets. Purulent fluid from a cecal ligation and puncture model, specific live and heat-killed bacteria (Escherichia coli, Salmonella typhimurium, and Listeria monocytogenes), and a variety of proinflammatory ligands (LPS, CpG DNA, Pam-3-Cys, flagellin, and TNF) were incubated with or without calf IAP (cIAP). Phosphate release was determined by using a malachite green assay. The various fluids were applied to target cells (THP-1, parent HT-29, and IAP-expressing HT-29 cells) and IL-8 secretion measured by ELISA. cIAP inhibited IL-8 induction by purulent fluid in THP-1 cells by >35% (P < 0.005). HT29-IAP cells had a reduced IL-8 response specifically to gram-negative bacteria; >90% reduction compared with parent cells (P < 0.005). cIAP had no effect on live bacteria but attenuated IL-8 induction by heat-killed bacteria by >40% (P < 0.005). cIAP exposure to LPS and CpG DNA caused phosphate release and reduced IL-8 in cell culture by >50% (P < 0.005). Flagellin exposure to cIAP also resulted in reduced IL-8 secretion by >40% (P < 0.005). In contrast, cIAP had no effect on TNF or Pam-3-Cys. The mechanism of IAP action appears to be through dephosphorylation of specific bacterial components, including LPS, CpG DNA, and flagellin, and not on live bacteria themselves. IAP likely targets these bacterially derived molecules in its role as a gut mucosal defense factor.
- Toll-like receptors
the gut mucosal barrier is a complex organ comprised of commensal microflora, a mucus layer, the epithelium, and the subepithelial immune system. A critical function of the gut barrier is to protect the host from both commensal and pathogenic luminal bacteria through control of inflammatory signaling and prevention of bacterial invasion/translocation. Of particular interest is the ability of the normal microflora to reside within the intestinal lumen without stimulating a pathological host immune response, as these same bacteria will cause severe inflammation and damage if they translocate across the gut barrier and spread to other organs. Breakdown of the gut mucosal barrier occurs in a variety of clinical conditions, including severe trauma and critical illness, often leading to systemic sepsis (7, 8, 33, 34, 51). Additionally, chronic inflammatory diseases such as Crohn's disease and ulcerative colitis are thought to be the result of a hypersensitive immune response to commensal bacterial components. In fact, the presence of commensal bacteria is a requirement for the development of inflammatory bowel diseases (39), and genetic mutations involving Toll-like receptor 4 (TLR4) and nucleotide-binding oligomerization domains 2/caspase recruitment domain 15 (NOD2/CARD15), both of which activate NF-κB in response to bacterial components, have been implicated in the pathogenesis of IBD (27, 42). As such, the interplay between the host and its commensal microflora is a topic of significant biological and clinical interest.
Intestinal alkaline phosphatase (IAP), a small intestinal brush border enzyme, appears to play a critical role in this host-microflora interaction. IAP is an alkaline phosphatase isoform that is produced exclusively in the small intestine and is both bound within the enterocytic cell membrane and secreted into the gut lumen (45). IAP has known definite physiological effects. It is thought to play a role in fat absorption, as demonstrated by studies showing that rat enterocytes induce IAP expression and secrete IAP-containing surfactant-like particles in response to fat feeding (31, 35). In addition, compared with their wild-type (WT) littermates, IAP knockout (KO) mice have been shown to become obese in the setting of a high-fat diet (36). However, recent data indicate that the more important physiological role for IAP relates to its function in regard to host gut mucosal defense.
In studies using zebrafish, Bates et al. (3) have shown that bacterial colonization of the intestinal tract induces IAP expression and, furthermore, that IAP is required for the normal recruitment of neutrophils to the gut. On the basis of these observations, the authors concluded that IAP is a key endogenous gut defense factor, functioning to maintain the critical homeostasis that exists between the host and the luminal microbial environment. Recent work from our laboratory has demonstrated that IAP-deficient mice (IAP-KO) suffer increased bacterial translocation into mesenteric lymph nodes when the gut was subjected to either direct (mesenteric ischemia) or remote (hind-limb ischemia) injury (21), further implicating IAP as an important gut defense factor. In addition to these studies on endogenous IAP function, exogenous administration of the IAP enzyme has been shown to attenuate the inflammatory effects, septic sequelae, and mortality from both LPS and gram-negative bacteria (6, 40, 50). IAP may also play an important role in regard to inflammatory bowel disease (IBD). Tuin et al. (49) examined IAP levels in human gut tissues and found a decreased expression in IBD patients, suggesting a possible causative role for IAP in the pathogenesis of human IBD. In addition, IAP treatment has been shown to be of benefit both in human ulcerative colitis patients and in a rat DSS-colitis model (29, 49).
These physiological and therapeutic effects of IAP have thus far been attributed to its known ability to dephosphorylate LPS (6). However, the precise mechanism by which IAP works remains unclear. It is uncertain whether IAP exerts direct effects on the bacteria themselves, or whether there are additional targets for IAP beyond LPS. Accordingly, the present studies were designed to more precisely define the molecular basis by which IAP functions as a gut mucosal defense factor. We demonstrate here that IAP acts as a gut mucosal defense factor with dephosphorylation and abrogation of inflammatory signaling by the specific bacterial components LPS, CpG DNA, and flagellin, and not by direct effects on bacteria themselves.
MATERIALS AND METHODS
WT mice (Mus musculus C57BL/6) were obtained from Jackson Laboratories (Bar Harbor, ME) and bred at the Massachusetts General Hospital (MGH) animal facility. The animal experiments were reviewed and approved by the IACUC at MGH. Animals were maintained in accordance with the guidelines of the Committee on Animals of Harvard Medical School (Boston, MA) and the Committee on the Care and Use of Laboratory Animals of the Institute of Laboratory Resources and the National Institutes of Health.
Cell culture and reagents.
Human colorectal adenocarcinoma HT29 cell lines were purchased from American Type Culture Collection (Manassas, VA) and maintained in McCoy's culture media (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS, Lonza, Allendale, PA), 2 mM l-glutamine and 100 units/ml each of streptomycin and penicillin (Invitrogen). HT29 cells were stably transfected with the IAP expression plasmid (HT29-IAP), and IAP enzyme activity confirmed as previously described (21). Human monocyte THP-1 cell lines were obtained as a generous gift from Dr. Hans-Christian Reinecker (MGH), and maintained in RPMI-1640 culture media (Invitrogen) supplemented with 10% heat-inactivated FBS, 2 mM l-glutamine, 100 units/ml each of streptomycin and penicillin, and 0.05 mM mercaptoethanol (Sigma, St. Louis, MO). LPS (Escherichia coli serotype 0111:B4, Sigma), TNF (Peprotech, Rocky Hill, NJ), CpG DNA (Fisher, Pittsburgh, PA), (S)-(2,3-bis(palmitoyloxy)-(2RS)-propyl)-N-palmitoyl-(R)-Cys-(S)-Ser(S)-Lys4-OH, trihydrochloride (Pam-3-Cys) (EMD Biosciences, Gibbstown, NJ), and flagellin (Salmonella typhimurium, Invivogen, San Diego, CA) were reconstituted, aliquoted and stored as directed. The CpG and flagellin are documented by the manufacturer as endotoxin “negative” (<0.125 EU/mg), and the products come with endotoxin-free water for reconstitution.
Bacterial culture and reagents.
E. coli and Listeria monocytogenes were clinical isolates from human (identified as 1554, 1555, and 1557) and mouse sources (1569). S. typhimurium was a gift from the Reinecker lab. Bacteria were maintained in culture with either Luria-Bertani or brain heart infusion media. When incubated with cell cultures, varying multiplicities of infection (MOI) and incubation times were investigated to obtain optimal IL-8 induction (noted in results).
Cecal ligation and puncture.
The cecal ligation and puncture model was utilized as a source of mixed infected fluid. WT mice underwent sedation with a mixture of inhalational nitrous oxide, oxygen, and 1.5% isoflurane. Midline abdominal incisions were made, the cecum ligated with a silk suture, and cecal puncture performed by using an 18-gauge needle. Abdominal incisions were subsequently closed, and the mice were allowed to recover and then euthanized after 24 h. The contents of the necrotic cecum were extracted and homogenized in sterile PBS. Optical densities (OD) were measured and the samples serially diluted. Varying concentrations of calf IAP (cIAP, 0–100 units, New England Biolabs, Ipswich, MA) were incubated along with the manufacturer's buffer, with 100 μl samples of varying dilutions of cecal contents for 2 h, then applied to THP-1 cells for 24 h. IL-8 secretion into cell culture media was measured by ELISA (BD Biosystems, San Diego, CA).
Malachite green assay.
LPS (1 mg), TNF (15 ng), CpG DNA (10 μM) and Pam-3-Cys (50 μg) were combined with cIAP (100 units) and buffer for 10 min to 24 h in sterile, endotoxin-free water. At predetermined time points, free phosphate released in solution was measured by using the malachite green assay kit per manufacturer's directions (BioAssay Systems, Hayward, CA). For bacterial incubations, 109 bacteria were centrifuged and resuspended in distilled endotoxin-free water, then incubated with cIAP (100 units/ml) for 2 h before assaying for free phosphate.
We chose IL-8 as a universal marker for inflammatory signaling, as IL-8 is released by many different cell cultures via diverse signaling pathways. HT29 cells were seeded into 12-well plates and grown until 60–70% confluency. LPS (0 to 10 μg/ml), TNF (0 to 15 ng/ml), and CpG DNA (0–2 μM) were incubated at 37°C with or without (+/−) cIAP (100 units/ml) and buffer for 2 h. The solutions were then directly applied to HT29 cells for 24 h. Media was collected and measured for IL-8. IL-8 induction by Pam-3-Cys (0–1 μg/ml) was measured in the same manner, but solutions were applied to THP-1 cells differentiated into macrophages by use of 2 nM phorbol-12-myristate-13-acetate (Sigma). Flagellin (0–100 ng/ml) was incubated +/−cIAP (400 units/ml) for 16 h and applied to undifferentiated THP-1 cells in 48-well plates for 24 h; supernatant was then harvested for IL-8 quantification.
Cell culture experiments were carried out in triplicate wells and results expressed as the average of values with one standard deviation. For the malachite green assay, results were averaged between duplicate or triplicate sample wells and expressed with standard deviation. All experiments were repeated a minimum of two times. Data presented here are of representative experiments. Statistical analysis between comparison groups was performed by using an unpaired Student's t-test.
IL-8 induced by a mixed purulent fluid is inhibited by cIAP.
The cecal ligation and puncture (CLP) model produces an intra-abdominal abscess that we used as the source for a mixed infected fluid, containing a variety of bacterial products and host inflammatory mediators. Even at very dilute concentrations, the infected fluid was routinely able to induce up to sixfold increases in IL-8 secretion by target THP-1 cells. However, when the CLP fluid was preincubated with cIAP, the effects on THP-1 cells were markedly inhibited. Inhibition was enzyme dose dependent; at 33 units of cIAP/cell culture well there was a >35% reduction in IL-8 secretion (P < 0.05, Fig. 1). This experiment demonstrated the ability of IAP to protect host cells from a mixed bacterial/inflammatory fluid. Given that the fluid obtained from the CLP model contains a complex mixture of bacteria and host mediators, we designed a series of experiments to identify the specific targets for IAP.
Expression of IAP in cell culture confers protection from the inflammatory response to gram-negative bacteria.
Parent and IAP-expressing HT29 cells were exposed to a variety of different enteric bacteria: a range of gram-negative bacteria, including E. coli strains isolated from human sources (MOI 100:1, incubation time 2 h), a mouse E. coli isolate (MOI 1,000:1, incubation time 2 h) and S. typhimurium (MOI 1,000:1, incubation time 2 h). We also chose L. monocytogenes, a gram-positive species that invades the intestinal tract and induces IL-8 secretion in intestinal epithelial cells (MOI 100:1, incubation time 3 h). Staphylococcus aureus and Enterococcus fecalis were tested as other gram-positive controls but did not provoke an inflammatory response in our parent cell line (Supplemental Fig. S1; supplemental material for this article is available online at the Journal website).
IAP expression by HT29 cells uniformly led to an inhibition in the IL-8 secretion that was induced by gram-negative bacteria (Fig. 2). While all gram-negative bacteria were able to induce an IL-8 response from HT29 cells, the response to mouse isolate E. coli 1569 and S. typhimurium were the most robust, with 10- and 14-fold increases in IL-8 levels, respectively (P < 0.005 vs. control, Fig. 2A). Human E. coli isolates 1554, 1555, and 1559 induced relatively modest 1.3-, 1.4-, and 1.7-fold increases in IL-8 secretion (P < 0.05, Fig. 2B). Compared with the results with parent cells, bacterial incubation with HT29-IAP cells resulted in much less IL-8 secretion, e.g., >90% reduction (P < 0.005) for E. coli 1569 and S. typhimurium (Fig. 2A). Inhibition of IL-8 induction by the human E. coli isolates was nearly complete (P < 0.05, Fig. 2B). Incubation with L. monocytogenes resulted in a 38-fold increase in IL-8 (P < 0.05 vs. control, Fig. 2C), which interestingly was not inhibited by IAP expression, indicating that the IAP effects were specific to gram-negative bacteria (Fig. 2C).
Exogenous IAP does not affect bacteria or bacterial stimulation.
Having established that cellular IAP expression confers protection against gram-negative bacteria, we next investigated the direct effects of the IAP enzyme on intact bacteria. First, E. coli 1569, S. typhimurium, and L. monocytogenes were incubated +/−cIAP (100 units/ml) and colony-forming units (CFUs) determined by plating aliquots at 0, 24, and 48 h. There was no difference in CFUs +/−cIAP at any time point (Supplemental Fig. S2), indicating that cIAP did not directly affect bacterial growth or viability. Given the known ability of IAP to dephosphorylate free LPS (20), we speculated that the enzyme might interact directly with the LPS within the bacterial cell walls of gram-negative bacteria. However, we could not detect any increase in free phosphate when E. coli 1569 or S. typhimurium were incubated with cIAP, suggesting that cIAP is not able to dephosphorylate the LPS contained within intact bacteria (Supplemental Fig. S3). These same bacteria were applied to HT29 cell cultures (2 h, 37°C) and IL-8 secretion assayed. Again, cIAP pretreatment had no effect on IL-8 induction. Furthermore, bacterial invasion was also unaffected by cIAP treatment (Supplemental Fig. S4). Taken together, these experiments indicate that the exogenous IAP enzyme is unable to directly alter the growth, surface biology or invasiveness of live, intact bacteria.
Exogneous IAP inhibits IL-8 secretion induced by heat-killed bacteria.
Having found that IAP expression within cell culture inhibited IL-8 induction by gram-negative bacteria, but also that exogenous IAP did not directly alter intact bacteria, we examined the effects of IAP on bacterial components. It is known that bacteria release proinflammatory components after invasion and after killing by antibiotics or heat (13, 14, 24, 25, 44). We therefore investigated whether the IAP enzyme would be able to alter the proinflammatory effects of heat-killed bacteria. Accordingly, E. coli, S. typhimurium, and L. monocytogenes (MOI 1,000:1) were heat killed by treating at 95°C for 30 min, incubated +/−cIAP (100 units/well cell culture) for 2 h, and then free phosphate was assayed. The heat-killed bacteria were next applied to HT29 cells for 3 h to assess their effects on IL-8 induction. Although cIAP incubated with live bacteria did not previously result in any demonstrable dephosphorylation, heat-killed bacteria treated with cIAP caused marked increases in free phosphate (Fig. 3A). Next, we found that the heat-killed bacteria were able to induce IL-8 production by HT29 cells (>100-fold increases over baseline, P < 0.05 for all three bacteria). In fact, heat-killed E. coli and L. monocytogenes were more proinflammatory than equivalent volumes of their live counterparts. Furthermore, in contrast to the results seen with intact, live bacteria, incubation of the heat-killed bacteria with cIAP caused a dramatic decrease in IL-8 induction (Fig. 3, B–D). There was >90 and 50% reductions (P < 0.005) for heat-killed E. coli and S. typhimurium, respectively, incubated with cIAP, and a 45% reduction for heat-killed L. monocytogenes (P < 0.005). These results with L. monocytogenes are particularly noteworthy in light of the fact that HT29-IAP cells were not earlier shown to inhibit IL-8 induction by this gram-positive bacteria, suggesting that LPS is not the only target for the IAP enzyme. Taken together, these results indicate that the heat killing of bacteria released one or more IAP targets and that these proinflammatory factors were previously unable to interact with the cIAP enzyme when present within an intact, live bacterium.
IAP specifically dephosphorylates the bacterial components LPS, CpG DNA, and flagellin, inhibiting their proinflammatory effects on target cells.
We next examined the effect of the IAP enzyme on specific bacterial components with regard to dephosphorylation and inflammatory signaling. The TLR ligands LPS (TLR4), CpG DNA (TLR9), flagellin (TLR5), and Pam-3-Cys (TLR2), as well as the nonbacterial ligand TNF, were chosen for study. It has been previously shown that both flagellin and LPS are dephosphorylated by cIAP, but it is not known whether IAP causes dephosphorylation of the other mediators (CpG, Pam-3-Cys, or TNF) (6, 26). As shown in Fig. 4B, incubation of CpG DNA with cIAP resulted in a twofold increase (P < 0.005) in free phosphate at just 30 min. Furthermore, absolute increases in the amount of released free phosphate rose over 24 h, indicating that cIAP-mediated dephosphorylation of CpG DNA is a time-dependent process (Fig. 5B). These dephosphorylating effects of cIAP on CpG DNA were similar to those seen with LPS, the previously described IAP target used for control purposes. In contrast, Pam-3-Cys and TNF incubation with cIAP did not result in any increase in free phosphate (Fig. 4, C and D), even after prolonged incubation times (Fig. 5, C and D).
Next, we investigated whether dephosphorylation of a particular ligand correlated with a decrement in its proinflammatory effects on target cells. LPS, CpG DNA, TNF, flagellin, and Pam-3-Cys all induced IL-8 secretion in HT29 cells or THP-1 cells in a dose-dependent manner. Preincubation of LPS, CpG DNA, and flagellin with cIAP resulted in >40% reductions (P < 0.005) in IL-8 secretion by target cells (Fig. 6, A–C). These inhibitory effects of cIAP were observed over a range of ligand concentrations. Furthermore, the inhibition of IL-8 production was cIAP dose-dependent (Supplemental Fig. S5). In contrast, IL-8 secretion induced by Pam-3-Cys and TNF in target cells was unchanged by cIAP (Figs. 6, D and E), demonstrating the specificity of IAP action for the particular target molecules, LPS, CpG DNA, and flagellin.
We also examined the time needed for the cIAP enzyme to alter the proinflammatory effects of a particular ligand. When LPS and cIAP were directly added to HT29 cells without preincubation, IL-8 was strongly induced. However, with 2 h of exposure to cIAP, LPS was unable to induce IL-8 secretion by the target cells (Supplemental Fig. S6). Finally, the effects of the various bacterial components were studied in HT29-IAP vs. parent cells. When LPS and CpG DNA, but not TNF, were incubated with HT29-IAP cells, IL-8 secretion was inhibited by >20% (P < 0.005) (Fig. 7). These results further support the concept that IAP alters the inflammatory response of host cells through an interaction with specific target ligands.
The intestinal brush border enzyme IAP has been long been recognized as an enterocyte differentiation marker, but its biological role has remained a mystery for decades. On the basis of recent work in both animals and humans, it appears that the major physiological function of endogenous IAP relates to its role in maintaining the critical homeostasis that exists between the host and the gut microbial environment. IAP appears to protect us from luminal microbes, inducing a controlled host inflammatory response within the gut and preventing bacterial translocation (3, 17, 21). In addition to this important role for the endogenous enzyme, IAP is showing great promise as a therapy for a variety of infectious and inflammatory conditions, including IBD and sepsis (6, 29, 49, 50).
Despite these many physiological and clinical observations, relatively little is known regarding the mechanisms underlying IAP action. The studies presented herein were undertaken to identify and better define the specific targets of action for the IAP enzyme. We first established a role for IAP in protection from septic mediators by showing that the enzyme has the ability to attenuate the inflammatory effects of contaminated fluid obtained from a CLP model. The series of experiments that followed were designed to systematically define the components of a mixed bacterial/inflammatory fluid that might be specific targets for IAP.
There were several considerations in our experimental design. IAP is a small intestinal brush border enzyme produced primarily in the proximal small intestine. Although IAP is contained within the cell membrane, the enzyme is also secreted into the intestinal lumen and, in fact, retains its activity throughout the length of small intestine and colon (21). One question addressed by the present studies is whether IAP conferred protection as an intracellular or extracellular factor. On the basis of our data, it is likely that IAP can exert its inhibitory functions both within and outside of the cell.
We first demonstrated that cellular IAP expression does inhibit inflammatory signaling induced specifically by gram-negative bacteria, but not by the gram-positive L. monocytogenes. The uniform inhibitory response to gram-negative bacteria by IAP expression in cell culture suggests that LPS is at least one of its specific targets, although the mechanisms by which bacteria induce IL-8 in intestinal epithelial cells are not all LPS dependent. For example, E. coli is known to induce IL-8 secretion in epithelial cells principally via the LPS-TLR4 pathway (2, 44), but the mechanisms for IL-8 secretion in the case of S. typhimurium are more complex, occurring through TLR5 and other pathways (15, 18–20). Interestingly, L. monocytogenes induces IL-8 by a non-TLR-dependent Nod pathway (38), and this signaling pathway was not inhibited by cellular IAP expression. In addition to the effects of cellular IAP, free, or exogenous, IAP also confers protection against specific inflammatory mediators, including LPS, CpG DNA, and flagellin. Further studies will be needed to directly prove that IAP inhibits these specific mediators in vivo and whether the effects occur in both the luminal and epithelial compartments.
Curiously, free IAP enzyme does not appear to directly affect live bacteria; cIAP has no effect on either bacterial viability or the ability of the intact bacteria to invade cells or induce a target cell inflammatory response. Rather, it appears that exogenous IAP functions to inhibit the inflammation induced by bacterial components, as when the bacteria were first heat-killed, incubation with cIAP abrogated the induction of IL-8 by target cells. Heat-killing of bacteria has been previously shown to enhance target cell cytokine production compared with live bacterial stimulation (16), a phenomenon thought to be caused by the release of proinflammatory bacterial components. A similar effect is observed clinically in the context of bacterial lysis by antibiotics, during systemic sepsis, and in the setting of meningitis (12, 14, 24, 25, 48). It is likely that the heat killing process releases LPS, bacterial DNA, flagellin, and perhaps other specific proinflammatory elements that are targets for the IAP enzyme. We speculate that the physical structure of intact live bacteria may not allow for a direct interaction between IAP and the bacterial components.
We were surprised to find that free phosphate was released when the gram-positive bacteria L. monocytogenes were heat-killed and incubated with cIAP. Furthermore, although HT29-IAP cells did not inhibit IL-8 induction by live L. monocytogenes, cytokine response was attenuated when heat-killed L. monocytogenes was preincubated with cIAP. These results suggested to us that IAP must target other non-LPS bacterial components capable of acting as proinflammatory mediators. On the basis of our data, it appears that, in addition to LPS, IAP also inhibits bacterial DNA and flagellin, two previously unrecognized targets for the IAP enzyme. It should be noted that the IAP effects were directly correlated with phosphate release. In the absence of dephosphorylation (i.e., TNF and Pam-3-Cys), there was no downregulation of the inflammatory pathway by IAP. In contrast, those inflammatory mediators that are dephosphorylated by IAP are the ones in which IL-8 secretion by the target cells was inhibited.
The fact that IAP dephosphorylation of LPS, CpG DNA and flagellin may alter their inflammatory effects is not very surprising. Toll-like receptors recognize pathogen-associated molecular patterns (PAMPs) by homology to specific structural conformations, and each TLR has a specific subset of associated PAMPs. For instance, LPS-TLR4 interaction depends on an intact lipid A (1), and it is well known that monophosphorylated LPS does not exhibit the same toxicity as intact endotoxin (5, 22). The role of dephosphorylation in the CpG DNA-TLR9 interaction has not been previously described, but it is known that ligand-receptor binding is specific to a sequence of oligodeoxynucleotides and is related to structural and conformational recognition (4, 32, 43). Dephosphorylation of the phosphodiester backbone terminal ends of CpG DNA residues might alter the structure or disrupt the precise CpG DNA sequence, leading to abrogation of ligand-receptor binding. In regard to the physiological importance of IAP targeting bacterial DNA, it is of interest to note that CpG DNA exposure worsens the inflammation in a mouse DSS colitis model (37). Given the beneficial role for IAP in regard to colitis as demonstrated in animals and humans (29, 49), we speculate that dephosphorylation of bacterial DNA by IAP may be one of its underlying mechanisms of action in this disease. Flagellin exerts its effects via TLR5, and recognition of the ligand is facilitated by conserved amino acid sequences (13, 26, 46). Flagellin tyrosine residues are posttranslationally modified by phosphorylation, which are then capable of being dephosphorylated by cIAP (26). It is possible that such dephosphorylation alters its interaction with TLR5, but this is not yet known. Further studies will be needed to precisely determine how IAP-induced dephosphorylation of bacterial DNA and flagellin could result in an inhibition of their proinflammatory effects. In addition, it should be recognized that the action of IAP on these bacterial ligands could also include mechanisms unrelated to dephosphorylation.
To fully understand the role of IAP as a defense factor against pathogenic bacteria, a larger number of specific bacterial inflammatory and invasive pathways will need to be examined. For instance, invasion by certain E. coli is dependent on LPS-TLR4 signaling (14, 44); yet invasion by S. typhimurium is largely unrelated to LPS. We have previously shown that starvation results in a specific loss of IAP expression in gut mucosa along with a decrease in intestinal dephosphorylating activity within the intestinal lumen (21, 23). In addition, the IAP-KO mice exhibit enhanced bacterial translocation under the stress of gut ischemia (21). Although a lack of IAP may render the intestinal mucosa more susceptible to inflammatory cytokine insults, the explanation for the increase in bacterial translocation is less clear. Further in vivo studies will be needed to determine how IAP modulation of specific TLR ligands affect various aspects of bacterially derived insults from commensal microbes and pathogens, including invasion, dissemination, and inflammation.
Of one last particular interest is the role that the TLRs play in maintaining intestinal flora homeostasis, allowing the colonization of the host with nonpathogenic bacteria while reducing their inflammatory impact. It has been suggested that commensal bacteria trigger the TLR signaling pathway to control intestinal epithelial homeostasis and protect the host from injury (28, 39), but in certain disease states such as Crohn's disease and necrotizing enterocolitis, TLRs (in particular, TLR4) are excessively upregulated (28). In considering the role of IAP in regard to gut inflammatory conditions, it may be that its effects occur both at the mucosal surface and within the luminal compartment. We speculate that endogenous antimicrobial peptides and other host factors kill or otherwise disable bacteria, resulting in the release of LPS, CpG DNA, and flagellin, molecules that subsequently are dephosphorylated and thereby inhibited by the IAP enzyme. It is likely that the interactions between IAP and these bacterially derived molecules play an important role in the intestinal homeostasis with commensal bacteria, as well as the host immune response to pathogenic bacteria.
This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants R01DK050623, R01DK047186, and T325T3DK007754.
No conflicts of interest, financial or otherwise, are declared by the author(s).
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