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From the Department of Pathology and Laboratory Medicine,* Epithelial Pathobiology Unit, Emory University School of Medicine, Atlanta, Georgia; and the Gut Immunology Group, Rowett Research Institute, Bucksburn, Aberdeen, United Kingdom
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Abstract |
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Ligand binding of all TLRs leads to initiation of several cytoplasmic signal transduction cascades that culminate with the appearance of activated transcription factors in the nucleus.5
These pathways include the Rel/nuclear factor (NF)-
B, the MAP kinase, and the IRF pathways, all of which exploit rapid, transient posttranslational modifications of protein intermediates to transmit signals to the nucleus. These alarm pathways stimulate the transcriptional up-regulation of a battery of proinflammatory effector proteins including chemokines, cytokines, and adhesion molecules. For example, previous expression profiling experiments from our and other laboratories demonstrated that purified flagellin interacting with its physiological receptor TLR5 was capable of initiating a proinflammatory transcriptional response almost identical to that induced by the canonical endogenous proinflammatory cytokine tumor necrosis factor-
.6-8
Regardless of the specific inciting ligand, up-regulation of proinflammatory effectors orchestrates circulating leukocyte recruitment and results in increased numbers of leukocytes entering affected tissues. It is generally accepted that whereas proinflammatory gene expression and the resulting acute inflammatory responses are necessary for the control/destruction of bacterial pathogens, this stereotypical tissue reaction is also causal of the clinical manifestations of enteric infection.9
Thus, MAMPs have been generally conceptualized as bacterial virulence factors—prokaryotic products that contribute to pathogenicity.
The Salmonellae are a group of gram-negative, facultative intracellular enteric pathogens causal of a spectrum of diseases in susceptible higher vertebrates.10 In human nontyphoidal Salmonella infection, the pathogen characteristically induces a classic acute proinflammatory response in infected intestinal mucosa.11,12 A combination of MAMPs and virulence factors translocated by type III secretion systems are known to contribute to the proinflammatory properties of this bacterium.13,14 We previously reported that flagellin is a key MAMP in epithelial responses to Salmonella infection in vitro. Mutation of the flagellin-encoding genes (FliC and FljB) in enteropathogenic S. typhimurium (SL3201) resulted in a viable and invasive microbe that was nearly devoid of proinflammatory activity at both the signaling and effector level.15 Importantly, we also showed that a mutant that secreted flagellin but was unable to assemble intact flagella (FliD) was fully proinflammatory, indicating that loss of motility did not account for the inability to stimulate inflammation. Thus, flagellin is necessary to induce inflammatory signaling and effector responses, the cardinal features of human disease, in model Salmonella infection of cultured epithelia.
To address the physiological role of flagellin-mediated proinflammatory signaling during infection in vivo, we have now used two distinct sets of isogenically matched wild-type (WT) and flagellin-deficient (aflagellate) Salmonella strains in two distinct murine models of enteric Salmonellosis. These isogenic strains presented the possibility of evaluating bacteria with and without a key MAMP, thus permitting loss-of-function analysis, an approach that is impossible with other MAMPs such as lipopolysaccharide and bacterial DNA that are required for viability. Unexpectedly, we observed that the aflagellate Salmonella mutants markedly increased disease severity. This aggravated clinical course corresponded to increased bacterial penetration from epithelia into systemic tissues. Importantly, enhanced bacterial translocation could be correlated with increased enterocyte apoptosis. These results expose an important cytoprotective component of TLR-mediated signaling induced during a bacterial-epithelial encounter and highlight its functional importance in the containment and control of gut-borne infections.
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Materials and Methods |
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Flagellin (FliC) from WT S. typhimurium (SL3201, fljB–) was purified through sequential cation and anion-exchange chromatography.16 Purity was verified as previously described.17 Antibodies used include cleaved caspase-3 (Cell Signaling Technology, Beverly, MA), RelA (Santa Cruz Biotechnology, Santa Cruz, CA), cIAP2, and anti-mouse lipocalin-2/NGAL (R&D Systems, Inc., Minneapolis, MN). Anti-ß-actin, human leukocyte myeloperoxidase (MPO), streptomycin, and hexa-decyltrimethylammonium bromide were from Sigma (St. Louis, MO). All other reagents were from Sigma unless otherwise stated.
Bacterial Strains Used
WT Salmonella enterica serovars typhimurium (SL3201) phase variable and its isogenic mutant (aflagellate, phenotype:nonmotile:genotype: fliC–/fljB–) were grown under microaerophilic conditions as previously described.15
Hereafter, these strains are referred to as WT-1 and aflagellate-1. FliD is a mutant in the flagellar hook protein and was a gift of Dr. R. Macnab (Yale University, New Haven, CT). WT Salmonella enterica serovars enteritidis (S1400/94) was originally isolated from a poultry infection and its isogenic mutant strain (aflagellate, phenotype:nonmotile:genotype: FliC::camR) was constructed by insertional mutagenesis.18
Hereafter these strains are referred to as WT-2 and aflagellate-2. Bacterial stocks were inoculated into 10 ml of Luria-Bertani broth and incubated with agitation at 37°C overnight to give 
109 CFU ml–1.
Murine Infections
Experimental Murine Enteritis
Pathogen-free female BALB/cj mice (6 to 8 weeks) were procured from Jackson Laboratories (Bar Harbor, ME). Animals were pretreated with streptomycin and then infected with Salmonella to induce colitis as described in Barthel and colleagues,19 with few modifications. On the day of the experiment, water and food were withdrawn 4 hours before treatment with 7.5 mg of streptomycin in 75 µl of sterile water by oral gavage. Afterward, animals were supplied with water and food ad libitum. At 20 hours after streptomycin treatment, water and food were withdrawn again for 4 hours before the mice were infected with 108 CFU of Salmonella typhimurium by gavage (WT-1, aflagellate-1, or FliD) and supplied with food and water. In some experiments, mice were pretreated with flagellin (20 µg/mouse i.p.) 2 hours before challenge with Salmonella. At the indicated times after infection, urine and blood were collected, and ceca were removed and fixed immediately in 10% buffered neutral formalin for histopathological analysis or thoroughly washed and snap-frozen for RNA isolation or homogenized for microbiological analysis by serial dilution on McConkey’s agar plates. Assessment of acute histological injury was based on the semiquantitative scheme used by Barthel and colleagues.19 For each experiment, five individual mice were evaluated for a given condition, scores totaled, and SE calculated by two independent blinded observers. Animal experiments were approved by the Emory University and Rowett Research Institute institutional ethical committee and performed according to the legal requirements.
Experimental Murine Natural Infection
Specific pathogen-free C3H/HeN female mice were used (5 to 6 weeks) (Harlan, Blackthorn, UK). Mice were given a single dose of LB broth (0.1 ml) containing 108 CFU S. enteritidis (WT-2 or aflagellate-2) by gavage. Food intake, body weights, and appearance/condition were monitored twice daily and scored according to the schedule of Shu and colleagues20 Tissues were flushed to remove contents and nonadherent bacteria, followed by weighing and homogenization. Viable bacteria were enumerated by serial dilution on XLD agar plates.
Ileal Loop
Green fluorescent protein (GFP)-labeled aflagellate-2 contained a GFP plasmid construct with an ampicillin resistance gene insert. The bacterial stock was maintained frozen and grown under the same conditions as the unlabeled strain, except that all media contained ampicillin (50 µg/ml). At 8 days after infection with unlabeled WT-2 or aflagellate-2, C3H/HeN mice were anesthetized (isoflurane), a ligated intestinal loop (2 to 8 cm from the ileocecal junction) prepared in situ, and GFP-aflagellate-2 (109 CFU) introduced. The mice were maintained under anesthesia at 35 to 37°C for up to 4 hours and then euthanized, and tissues removed for analysis.
Tissue Myeloperoxidase Activity
Neutrophil infiltration into tissue was quantified by measuring MPO enzyme activity (a marker for neutrophils). Briefly, after thorough washings to remove the contents, tissue (50 mg/ml) was homogenized in 0.5% hexadecyltrimethyl ammonium bromide (in 50 mmol/L phosphate buffer, pH 6.0), freeze-thawed three times, sonicated, and centrifuged. MPO was assayed in the clear supernatant (14 µl) by adding 1 mg/ml dianisidine dihydrochloride and 5 x 10–4% of H2O2 and the change in optical density measured at 450 nm. One U of MPO activity was defined as the amount that degraded 1.0 µmol of peroxide/minute at 25°C.21
Serum Cytokine Assays
Mice were bled at 6, 12, 24, and 48 hours after infection through the retro-orbital plexus, and serum was isolated by centrifugation. Serum KC and IL-6 assays were performed using mouse Duoset enzyme-linked immunosorbent assay kits (R&D Systems) according to the manufacturer’s instructions. Serum amyloid A was measured by enzyme-linked immunosorbent assay kit (sensitivity 10 ng/ml) procured from Bender MedSystems (Burlingame, CA) and Biosource (Camarillo, CA).
Cell Culture
Primary rat intestinal epithelial cells (IEC-6) and CaCo2 cells were maintained as described.6 Monolayers were washed twice with Hank’s balanced salt solution at 37°C and equilibrated for 15 minutes before treatment. For bacterial treatments, bacterial cultures were washed, concentrated, and applied to the apical aspects of cells at an multiplicity of infection of 30 as described.15
Immunoblotting
For lipocalin-2 immunoblot assessment, serum and urine samples were diluted 1:10 and 1:2, respectively, in loading buffer, subjected to 4 to 20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting, and probed with biotinylated anti-mouse-lipocalin-2/NGAL monoclonal antibody (0.2 µg/ml). For immunoblot of cell lysates, cells were lysed in ice-cold buffer containing 50 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and protease inhibitor cocktail (Roche Diagnostics Corp., Indianapolis, IN). Immunoblot was performed as described.6
Electrophoretic Mobility Shift Assay of NF-B
Nuclear extracts from untreated and treated cells were incubated with double-stranded 32P-labeled oli-gonucleotide probes containing consensus-binding sequences for NF-B (5'-AGTTGAGGGGACTTTCCCAGGC-3') separated by electrophoresis and visualized by autoradiography.
Real-Time Quantitative Polymerase Chain Reaction (PCR)
Total RNA from mouse cecum were prepared using TRIzol reagent (Invitrogen, Carlsbad, CA). Reverse transcriptase (RT)-PCR was preformed as described.6 Primers for the genes of interest were designed with PrimerExpress (Applied Biosystems, Foster City, CA), and their sequences are available on request.
Fluorogenic Caspase Staining
IEC-6 cells were grown on glass coverslips as described earlier. Bacterial suspensions were distributed as 400-µl beads over a piece of parafilm, with the coverslip applied in an inverted manner. This experimental design was to eliminate confounding results potentially caused by settling and accumulation of immotile mutants. Caspase-8, -9, and -3 activation was detected using the APO LOGIX carboxyfluorescein caspase detection kit (Cell Technology, Mountain View, CA). Positive cells were visualized by confocal microscopy (Zeiss LSM 510) at 505 nm.
Cell Death Assay
IEC-6 cells after treatment (floating and adherent), were collected at various times for terminal dUTP nick-end labeling (TUNEL) staining; the In Situ Cell Death Detection Kit (Roche Diagnostics Corp.) was used according to the manufacturer’s instructions. In brief, IEC-6 cells were harvested and fixed in 2% paraformaldehyde, permeabilized in 0.1% Triton X-100 in 0.1% sodium citrate, and labeled with TUNEL reaction mixture. For morphological evaluation of TUNEL positivity, cells were co-cultured with bacteria in an inverted manner as described above and visualized by confocal microscopy (Zeiss LSM 510) at 505 nm. For analysis of bacteria-induced apoptosis by flow cytometry, 10,000 fluorescent events were measured for each sample by a FACScalibur flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA) using the Cell Quest software. Data were analyzed using FlowJo software.
Immunofluorescence Analysis
After experimental treatments CaCo2 cells grown on 35-mm culture dishes were fixed in 4% paraformaldehyde or methanol and permeabilized in 0.2% Triton X-100/phosphate-buffered saline. Cells were stained as previously described.17
Statistical Analysis
The statistical method was Student’s t-test with either one-tailed or two-tailed distribution as appropriate.
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Results |
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To better understand the role of flagellin in Salmonella infections in vivo, WT Salmonella strains were compared with isogenic aflagellate mutants in an acute model of Salmonella enteritis. Recently, Hardt and colleagues19,22-24
showed that clinical isolates of S. typhimurium, which normally cause little intestinal inflammation in mice, will induce a robust neutrophilic colitis highly reminiscent of human Salmonellosis when mice are pretreated with streptomycin. We infected BALB/c mice in this manner using a WT S. typhimurium clinical isolate (SL3201—designated WT-1) and an isogenic mutant lacking both flagellin genes (SL3201—fliC–/fljB– designated aflagellate-1). We observed that mice infected with WT-1 developed a watery diarrhea within 12 hours after colonization. The incidence of diarrhea was significantly less in response to infection with an isogenic aflagellate mutant (incidence of diarrhea was 60 versus 20% in response to WT-1 and aflagellate-1 mutant, respectively; P < 0.05). Moreover, histopathological analysis showed that mice infected with WT-1, but not the aflagellate-1, exhibited neutrophil infiltration accompanied by mild submucosal edema as early as 6 to 12 hours after infection (Figure 1A)
. These histological changes closely correlated with tissue MPO (Figure 1B)
and tissue injury score (Figure 1C)
, which increased within this time frame only in response to the WT-1 strain. The elicitation of a strong rapid inflammatory response by the WT-1, but not aflagellate-1, strain was not only evident by examining intestinal parameters but was also reflected systemically with elevated serum keratinocyte-derived chemokine (KC-murine homolog of IL-8) (Figure 1D)
, serum levels of lipocalin-2/NGAL (Figure 1F)
, and serum amyloid A [known to represent a general marker of acute inflammation (Supplementary Figure 1A
at http://ajp.amjpathol.org)].25,26
Similarly, urinary excretion of lipocalin was significantly higher in mice challenged with WT-1 (Supplementary Figure 1B
at http://ajp.amjpathol.org). Thus, consistent with in vitro results that indicate flagellin is a dominant proinflammatory determinant of enteropathogenic Salmonella, aflagellate S. typhimurium induced markedly less acute inflammation in a murine model of Salmonellosis.
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, top; and Supplementary Figure 1C at http://ajp.amjpathol.org). This gross phenotype was observed in 13 and 87%, respectively, of mice infected with WT-1 or aflagellate-1, P < 0.05. In addition, cecal histopathology induced by aflagellate bacteria was more severe with increased numbers of crypt abscesses as well as massive edema (Figure 2A
, bottom), again highly correlated with increased mucosal MPO (Figure 1B)
and tissue response (Figure 1C)
. At these time points, aflagellate-1 infection also stimulated greater systemic markers of inflammation: serum KC, IL-6, and lipocalin-2/NGAL (Figure 1, D–F)
. Furthermore, at 48 hours, mice challenged with aflagellate-1 exhibited significantly higher numbers of bacteria adherent to or within the cecal mucosa, relative to mice infected with WT-1 (Figure 2B)
, confirming the infectivity of aflagellate-1 and suggesting reduced bacterial clearance. Thus, by 48 hours after infection, as compared with WT-1, aflagellate-1 resulted in a more severe disease process.
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, and Figure 2A
), indicating that the enhanced clinical effects of the aflagellate Salmonella were not the result of immotility of the bacteria. Aflagellate Salmonella Are Hypervirulent in Vivo in C3H/HeN Mice
We next studied a model of enteric Salmonellosis based on oral infection in C3H/HeN mice, which, unlike many inbred mouse strains, have isoforms of the gene Slc11a1+ that are associated with relatively high natural resistance to Salmonella. To address the role of flagellin in this form of enteric Salmonellosis, we again compared the outcome of infection with a WT Salmonella enterica serovar enteritidis isolate (designated WT-2) or its isogenic aflagellate mutant fliC:camR derivative (designated aflagellate-2). C3H/HeN mice were infected with a single dose of 108 organisms and monitored for up to 12 days after infection. Bacterial colonization was prevalent in ileal, cecal, and colonic tissues (Table 1)
and was accompanied by crypt hyperplasia, edema of submucosa, and PMN infiltration into the lamina propria and crypts (Figure 3A, i–iv)
. No differences in intestinal colonization were seen with the WT-2 or aflagellate-2 Salmonella at 6 and 12 days. Systemic infection, quantified by the numbers of bacteria recovered from spleen and liver, was at a low level at 6 days after infection (Table 1)
. However, by 12 days after infection the numbers of bacteria found in peripheral organs, including extra-reticuloendothelial tissues such as kidney, was significantly greater in aflagellate-2-infected mice (Table 1)
and correlated with a more severe disease manifested by greater weight loss (data not shown) and increased ileal MPO levels (Figure 3B)
. In aflagellate-2-infected mice the liver and spleen weights were significantly higher than in WT-2-infected mice, indicative of greater bacterial infiltration of these reticuloendothelial tissues (Supplementary Figure 2, A and B
, at http://ajp.amjpathol.org). Ultimately, greater mortality was observed with aflagellate-2 infection, which was particularly evident in younger/smaller mice (Figure 3C)
. Thus, in two independent murine models of enteric Salmonellosis, aflagellate bacteria caused a more severe clinical response.
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] of mice infected with aflagellate-2. This enhanced transepithelial translocation correlated directly with the greater systemic bacterial load observed in aflagellate-2-infected mice. Furthermore, as both the number of GFP bacteria in the gut lumen and/or those closely adhered to the gut tissues were not significantly different between WT-2 and aflagellate-2-infected mice (data not shown), it is likely that enhanced uptake and/or break-out from the gut tissues into submucosal/systemic tissues explains the elevated systemic infection in aflagellate-infected mice.
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Our data show that the absence of flagellin on a viable pathogen resulted in a more severe clinical phenotype. We next sought to determine whether the tissue effects could be reversed or attenuated by administration of purified flagellin monomers, the physiological ligand that activates TLR5. We observed that purified flagellin given systemically (20 µg/mouse i.p.) 2 hours before oral inoculation of the aflagellate-1 could attenuate host tissue response, as assessed by gross and histopathological parameters, to levels associated with infection with WT-1 bacteria (Figure 5A, i and ii
; Supplementary Figure 1C at http://ajp.amjpathol.org). Consistently, cecal MPO and serum KC levels at 48 hours after infection were significantly reduced after exogenous flagellin pretreatment (Figure 5B, i and ii)
. Furthermore, exogenous flagellin was able to substantially reduce the recovered bacterial load (Figure 5B, iii)
. In addition, we found that flagellin pretreatment could reduce the level of systemic KC and intestinal MPO induced by WT-1 flagellated Salmonella (Figure 5B, i and ii)
. These data indicate that the proinflammatory responses to flagellin are not maximal during the infectious process and can be further attenuated with additional flagellin. Given the widely observed ability of flagellin-induced gene remodeling in intestinal epithelial cells,27,28
this result suggests that the reversion of histological injury by flagellin is the consequence of TLR5-mediated transcriptional responses.
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In the C3H/HeN model with aflagellate S. enteritidis, evaluation of intestinal epithelia throughout the course of the infection did not reveal clear-cut morphological differences in cellular inflammation between the WT-2-infected mice (although MPO levels were significantly higher in aflagellate-2-infected mice at 12 days after infection, implying a greater neutrophilic infiltrate; Figure 3B
). However, strikingly increased numbers of apoptotic bodies were observed within ileal epithelia infected with the aflagellate-2 (Figure 6A, i and ii)
. Next, we sought to determine whether increased epithelial apoptosis was occurring within the time frame of the acute enteritis model using BALB/cj mice and S. typhimurium. Immunohistochemical stain for activated caspase-3 showed a markedly greater amount of immunoreactivity in the cecal tissues of mice infected with aflagellate-1 bacteria relative to infection with WT-1 at 12 hours after infection, the time point before increased inflammation was observed (Figure 6B, i–iii)
. Denudation and/or epithelial erosions were not observed. Apoptosis is well known as an intrinsic process by which individual cells can be eliminated while sparing overall tissue integrity,29
and caspase activation is established as an inhibitor of inflammatory pathways.30,31
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, suggesting that diminished proinflammatory signaling resulted in a proapoptotic state in vivo. Aflagellate Salmonella Induces Increased Apoptosis in Cultured Epithelial Cells
We next sought to compare apoptotic activation elicited by flagellate WT-1 and isogenic aflagellate-1 in the context of model infection in cultured primary epithelial cells. Epithelial monolayers were infected with bacteria at a multiplicity of infection of 30, a ratio that extensive past work has shown to mediate maximal signaling and transcriptional activation.16,17,32
Caspase activation of the extrinsic pathway (caspase-8) occurred within hours in cells infected with WT-1; however, the aflagellate-1 revealed a markedly increased degree and number of positive cells, with faster kinetics. Likewise, activation of caspase-9 (intrinsic pathway) and caspase-3 (executioner pathway) were slightly delayed but showed an identical acceleration and intensification when infected with aflagellate-1 (Figure 7A)
.
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. Aflagellate Salmonella Fails to Induce Survival Pathways and Effector Genes in Cultured Epithelial Cells
We have demonstrated that purified flagellin can activate a NF-B-responsive reporter gene and that blockade of NF-B, either pharmacologically with proteasome inhibitors or with a dominant-negative IB construct, potentiates epithelial cell apoptosis, suggesting that flagellin-induced NF-B can function as an important anti-apoptotic survival pathway.6
To determine whether aflagellate bacteria were proapoptotic because of the failure to induce the NF-B pathway, we infected cells and assayed for NF-B activation. As expected, and consistent with previously published data, WT bacteria induced both NF-B nuclear translocation and DNA binding during a 4-hour infection (Figure 7, C and D)
. In contrast, the aflagellate mutants showed a greatly reduced induction of this signaling pathway in both assays.
WT Salmonella infection is a potent transcriptional inducer of well-known anti-apoptotic factors.6
We tested if aflagellate mutants fail to up-regulate these cytoprotective genes in model epithelium, as suggested by in vivo data (Figure 6C)
. In immunoblots for the highly expressed anti-apoptotic effector cIAP-2, we demonstrated that aflagellate-1 is a weaker inducer of this protein, and this decreased expression corresponds with enhanced activation of executioner caspase-3 (Figure 7E)
. These observations are consistent with the inability of these bacteria to activate the NF-B and MAPK pathways and, thus, the anti-apoptotic proteins that normally serve to inactivate caspases.15
Flagellin-induced survival gene expression may protect cells from concurrent and/or subsequent proapoptotic signaling. We show that in vivo administration of flagellin attenuates clinical and histopathological effects of infection with aflagellate Salmonella (Figure 5)
. To determine whether this treatment could attenuate apoptotic signaling in vitro, IEC-6 cells were pretreated with purified flagellin before a proinflammatory stimulus, ie, aflagellate-1 mutant. Previous work has shown that maximal induction of anti-apoptotic gene expression occurs within 4 hours of flagellin stimulation.6
Cells were then challenged with aflagellate-1 for 6 hours, and caspase activation was quantified with luminescent substrates for executioner caspase-3. As shown in Figure 7F
, flagellin pretreatment reduced epithelial apoptosis elicited by aflagellate-1, relative to caspase induced in untreated cells.
Collectively, from this series of in vivo and in vitro experiments, we conclude that in loss-of-function experiments, aflagellate bacteria are able to elicit increased caspase activation and apoptosis, and in gain-of-function experiments, this caspase activation can be inhibited by exogenous flagellin. Aflagellate bacteria do not activate the NF-B pathway and only weakly induce survival genes; hence, concurrent apoptotic stimuli are able to proceed unimpeded. Perception of flagellin present on invading flagellated Salmonella by the host epithelial cells, and consequent inhibition of epithelial cell apoptosis may be an important mechanism limiting both the translocation of invading bacteria from epithelial tissues to the lamina propria and systemic tissues as well as the tissue pathology associated with infection-driven inflammation.
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Discussion |
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). Plausibly, the failure of aflagellate Salmonella to stimulate early proinflammatory and/or antibacterial gene expression permits these bacteria to delay innate immune detection and establish a foothold within the gut mucosa, thereby promoting their ability to gain access to the lamina propria and resident and/or newly recruited immune cells therein. In parallel, the failure of epithelial cells to respond to aflagellate bacteria with anti-apoptotic and other aspects of cytoprotective signaling accelerates caspase activation/apoptosis that further compromises the anti-microbial response and potentially facilitates bacterial release to the subepithelial tissues. Consistent with this latter suggestion, recent data have shown that TLR/MyD88 signaling is an important pathway limiting bacterial translocation to the lamina propria and, hence, susceptibility to systemic Salmonellosis.24
Thus, we conclude that flagellin-induced signaling plays a previously unappreciated role in infectious disease pathogenesis, orchestrating not only well-characterized recruitment of phagocytes but also cytoprotective effects that ultimately limits the apoptotic response of intestinal epithelial cells.
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Differences in the pathogenesis of infection with WT and aflagellate Salmonella have been described previously. Stecher and colleagues37 evaluated a nonflagellated S. typhimurium mutant (fliGHI) in the streptomycin pretreatment model system in C57BL6 mice, reporting that aflagellate organisms efficiently colonized the cecum by 10 hours, showed increased systemic dissemination, and, highly consistent with our results, showed that these mutants elicited markedly less cellular inflammation and histologically demonstrable tissue injury throughout the first 48 hours after infection. These experiments did not examine longer time points nor apoptosis.37 Schmitt and colleagues38 evaluated survival in mice (also highly susceptible C57BL6) infected with S. typhimurium bearing a FljB-null genotype, as well as the FliC/FljB mutants used in our study, finding a modest increase in mortality. In a chicken model, an aflagellate S. typhimurium mutant (FliM) showed an enhanced ability to establish a systemic infection. Also consistent with our findings, this strain elicited reduced IL-1ß up-regulation and PMN infiltration into the gut in early stages of infection.39 Interestingly, flagellin perception is important in containment of bacterial dissemination in pathogen-plant interactions.40 Li and colleagues41 reported that flagellated Pseudomonas syringea activated specific nonhost responses in Arabidopsis, whereas aflagellate mutants induced markedly weaker responses and, as a consequence, proliferated within the plant to a greater extent and ultimately caused a more severe disease.
Apoptosis and Inflammation in Enteric Infection
Apoptosis, or programmed cell death, is a morphologically distinct, genetically defined intrinsic mechanism by which individual cells can eliminate themselves while primarily preserving the surrounding cells.29,42 Apoptosis is a common cellular response to bacterial infection, playing a key role in the pathogenesis of bacterial infections such as Yersinial plague and systemic anthrax, in which the target cell is the macrophage.43 Epithelial apoptosis induced by bacteria has also been implicated in the pathogenesis of enteric infection.44 Extensive mucosal apoptosis and epithelial damage has been described in experimental and clinical cases of Shigella infection.45,46 Intriguingly, Shigellae are aflagellate pathogens. Potentially, this aggravated clinical picture may be mediated by lack of ligands such as flagellin that play an important role in mediating cytoprotective responses. Salmonella are potent inducers of apoptosis in macrophages,47 although the role of apoptosis in epithelial-Salmonella interactions has only been described in vitro. Previously, we have shown that a type III secreted effector of Salmonella, AvrA, could induce apoptosis when overexpressed in epithelial cells.48 Kagnoff and colleagues33,49 have shown in Salmonella epithelial co-culture experiments that viable organisms induced apoptosis after a surprisingly prolonged interval of 12 to 18 hours and suggested that delayed epithelial apoptosis provides sufficient time for the epithelia to generate survival/defensive responses, as well as a safe interval for bacteria to up-regulate genes (ie, SPI-2) necessary for survival in the intracellular environment. The end result of this highly evolved equilibrium between host and pathogen is self-limited enteritis. Our data indicate that flagellin-mediated signaling accounts for this apoptotic delay via activation of survival factors. In the absence of flagellin, the usual equilibrium is disturbed, and suppression of apoptosis does not occur. Apoptotic activation is well known to repress proinflammatory signaling via caspase-mediated degradation of signaling intermediates such as IKKß and p65.30,31,50 Again, the failure to control intracellular caspase activity would be expected to paralyze the early proinflammatory response of the gut epithelia during the early stages of infection, thus allowing bacterial proliferation and unchecked bacterial invasion/dissemination to subepithelial and systemic tissues.
Studies of classic inflammatory responses clearly show that a subset of the acute inflammatory effectors includes anti-apoptotic survival proteins such as cIAP1, cIAP2, cFLIP, and A20.50-52
NF-B-dependent transcriptional up-regulation of these proteins arrests any concurrent caspase activation, allowing inflammation without cell death. This interrelationship was demonstrated in the intestine by Karin and colleagues53
using mice conditionally null in intestinal epithelial NF-B, which when challenged with a local hypoxic challenge, triggered massive epithelial apoptosis. In this case, the epithelium was constitutively unable to activate prosurvival pathways when confronted with an environmental stress (hypoxia). These events explain the apoptosis commonly recognized during pharmacological inhibition of NF-B or when this pathway is blocked by the action of specific bacterial effector proteins.54
Aflagellate Salmonella (indeed any invasive Salmonella and many other pathogens) are able to initiate caspase activation in the epithelial barrier, likely because of type III effectors, secreted toxins, metabolic stress, and so forth, but the lack of a proinflammatory response, necessarily including survival gene up-regulation, permits progression to excessive local apoptosis. Our data suggest MAMP-TLR interactions may have an important role in activation of survival pathways in vivo, which results in attenuating virulence in certain infections.
Cytoprotective Effects of Exogenous Flagellin
Cytoprotection denotes a given agent or gene product that can reduce apoptosis or necrotic cell death (either in cell culture or in vivo) in response to a stressor. An emerging idea in intestinal biology is that TLR signaling can in fact be cytoprotective.27,55 Rakoff-Nahoum and colleagues56 demonstrated that mice with intestines cleared of normal flora, and thus MAMPs, were markedly more sensitive to the chemical colitogen dextran sodium sulfate and that the mucosal injury mediated by this compound could be ameliorated by oral administration of MAMPs such as lipoteichoic acid and lipopolysaccharide. In addition, this study demonstrated that these protective effects were lost in TLR2- and TLR4-null mice, implicating TLR signaling as the protective mechanism. Several genes were identified as candidate protective genes, although known anti-apoptotic effectors were not described in this study. Consistently, we have demonstrated that administration of flagellin and poly(I:C) (a synthetic double-stranded RNA and ligand for TLR3) can also ameliorate dextran sodium sulfate colitis (M. Vijay-Kumar and A.T. Gewirtz, submitted).
Beneficial effects of probiotic bacteria can be recapitulated by isolated MAMPs. For example, unmethylated probiotic CpG DNA has been shown to ameliorate dextran sodium sulfate colitis. Furthermore, the observed protective effects were lost in TLR9-null mice, directly implicating TLR signaling in intestinal cytoprotection.57,58 TLR signaling from MAMPs has also been shown to directly inhibit apoptotic processes in neutrophils.59 Other MAMP-inducible genes such as stromal growth factors and angiogenic factors have reparative functions, and may be protective in the sense of accelerating restitution. Interestingly, flagellin, but not other MAMPs, can induce cell cycle entry and proliferation in cultured fibroblasts.60 In addition to directly activating anti-apoptotic gene expression, flagellin may also activate a variety of other cytoprotective mechanisms (eg, heat-shock proteins, trefoil factors) whose actions might further, indirectly, reduce epithelial cell apoptosis. Taken together a picture is emerging that the mucosa—epithelial cells, immunocompetent resident cells in the lamina propria, or both—can perceive the abundant MAMPs in the intestinal lumen and induce a transcriptional response resulting in enhanced cyto-defenses. Potentially, luminal administration of TLR ligands such as flagellin may induce cytoprotective responses in this situation and act as a palliative.
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Acknowledgements |
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Footnotes |
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Supported by the National Institutes of Health (RO1 grants AI51282, DK071604 to A.S.N. and DK061417 to A.T.G.), the Emory Digestive Disease Research Center (R24 DK064399), the Crohn’s and Colitis Foundation of America (Senior Investigator Award to A.S.N.), and the Scottish Executive for Environment and Rural Affairs Department (to D.K.).
M.V.K. and H.W. share primary authorship and A.T.G. and A.S.N. share senior authorship.
A.S.N. is a Burroughs Wellcome Investigator in the Pathogenesis of Infectious Disease. M.V.K. and R.J. are Crohn’s and Colitis Foundation of America Research Fellowship recipients.
Supplementary materials for this article can be found on http://ajp.amjpathol.org.
Accepted for publication July 20, 2006.
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  M. Vijay-Kumar, J. D. Aitken, C. J. Sanders, A. Frias, V. M. Sloane, J. Xu, A. S. Neish, M. Rojas, and A. T. Gewirtz Flagellin Treatment Protects against Chemicals, Bacteria, Viruses, and Radiation J. Immunol., June 15, 2008; 180(12): 8280 - 8285. [Abstract] [Full Text] [PDF]
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 M. Vijay-Kumar, J. D. Aitken, A. Kumar, A. S. Neish, S. Uematsu, S. Akira, and A. T. Gewirtz Toll-Like Receptor 5-Deficient Mice Have Dysregulated Intestinal Gene Expression and Nonspecific Resistance to Salmonella-Induced Typhoid-Like Disease Infect. Immun., March 1, 2008; 76(3): 1276 - 1281. [Abstract] [Full Text] [PDF]
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A. Kumar, L. D. Hazlett, and F.-S. X. Yu Flagellin Suppresses the Inflammatory Response and Enhances Bacterial Clearance in a Murine Model of Pseudomonas aeruginosa Keratitis Infect. Immun., January 1, 2008; 76(1): 89 - 96. [Abstract] [Full Text] |
