Salmonella typhimurium translocates flagellin across intestinal epithelia, inducing a proinflammatory response (original) (raw)
S. typhimurium–epithelial interactions result in the release of a proinflammatory protein. S. typhimurium contact with the apical surface of model intestinal epithelia induces an increase in the epithelial intracellular [Ca++] that leads to activation of the transcription factor NF-κB and subsequently synthesis and secretion of IL-8 (7). The latter (i.e., NF-κB activation, IL-8 secretion), but not proximal Ca++-mediated portion of this signaling pathway, is shared by the proinflammatory cytokine TNF-α. This led to the hypothesis that the [Ca++] increase induced by S. typhimurium could cause the epithelial exocytosis of a proinflammatory mediator that could then activate IL-8 expression by a mechanism similar (or identical) to that used by TNF-α. Thus, we collected the conditioned media of _S. typhimurium_–infected model epithelia 1 hour after application of the bacteria and examined the ability of these samples to activate IL-8 secretion in virgin (i.e., noninfected) epithelia. After filtration through a 0.2-μm pore filter to remove bacteria, the apically conditioned media was transferred to the apical surface of virgin epithelia while the basolaterally conditioned media was transferred to the basolateral aspect of an additional virgin epithelia. Five hours later, we measured the IL-8 concentration in the basolateral reservoir because IL-8 secretion is known to a occur in a basolaterally polarized manner regardless of the type or location of stimulus (23). As shown in Figure 1a, transfer of basolaterally conditioned media, but not apically conditioned media, significantly induced IL-8 secretion, indicating the existence of a PIF. Nonphysiological transfer of apically conditioned media to the basolateral surface of virgin epithelia, but not transfer of basolaterally conditioned media to the apical surface, also induced IL-8 secretion (data not shown), suggesting the response to PIF, rather than the release of PIF, was polarized (addressed later in this article). PIF bioactivity (i.e., ability to induce IL-8 secretion) became detectable in the basolateral media 15 minutes after apical addition of the bacteria, and the amount of PIF bioactivity increased thereafter (Figure 1b). Induction of IL-8 secretion was henceforth used as a measure of PIF bioactivity.
Basolaterally conditioned media of _S. typhimurium_–infected epithelia induce IL-8 secretion in virgin epithelia. Model intestinal epithelia were apically exposed to S. typhimurium. The epithelial media was then transferred to untreated model epithelia. IL-8 was assayed in basolateral media 5 hours later. (a) Media was transferred maintaining its polarity of origin (i.e., apical to apical, basolateral to basolateral) 1 hour after addition of bacteria. Live S. typhimurium (added apically for 5 hours) served as a positive control. (b) Basolateral media was transferred at the indicated time after addition of bacteria.
We next began a biochemical characterization of PIF. To obtain an approximate size estimation of PIF, we filtered it using molecular weight (MW) cut-off filters. PIF activity was completely retained in the retentate of 50 kDa (and lower) filters and variably retained by higher MW cut-off filters (Figure 2a). Next, we measured PIF sensitivity to 20 minutes of boiling. This treatment did not reduce the ability of PIF to induce IL-8 secretion in model epithelia (i.e., PIF bioactivity) (Figure 2b). In contrast, heating TNF-α in this manner resulted in a complete loss of its bioactivity. We next measured the effect of treating PIF with proteinase K. PIF bioactivity was completely destroyed by this treatment (Figure 2b), indicating that PIF contains an essential protein component. To ensure that the proteinase K was not interfering with the IL-8 induction assay (even though the proteinase K was inactivated by boiling), we verified that this proteinase had no effect on IL-8 induction by carbachol. Lastly, we verified that PIF behavior regarding MW cut-off filters did not differ if PIF was first boiled before being applied to these filters (data not shown), suggesting that PIF was indeed a fairly large molecule rather than a smaller molecule being bound by a larger one. Together, these results indicate that PIF is a heat-stable protein with an approximate MW of 50–100 kDa.
PIF is a heat-stable protein. Samples from a and b were applied basolaterally to model epithelia, and their ability to induce IL-8 secretion over a 5-hour period was measured. (a) PIF was centrifuged through indicated Amicon concentrators followed by dilution of retentate to original volume. (b) PIF, TNF-α (20 ng/ml), and carbachol (100 μM) were subjected to boiling (20 minutes) or proteinase K treatment (1 mg/ml for 1 hour followed by 10 minutes of boiling to inactivate proteinase K).
PIF release is not induced by nonbacterial agonists. To investigate whether PIF was of epithelial, rather than bacterial, origin we attempted (unsuccessfully) to induce epithelial PIF release with nonbacterial agonists. Basolaterally conditioned media of TNF-α–treated model epithelia were isolated 45 minutes after addition of this agonist. These samples were then boiled to inactivate the TNF-α without harming any PIF that may have been secreted. Samples isolated in this manner did not display any IL-8–inducing bioactivity (Figure 3), indicating that TNF-α does not induce the release of PIF. Next, we investigated whether PIF release could be induced by the acetylcholine receptor agonist carbachol in accordance with our hypothesis that PIF is secreted by Ca++-mediated exocytosis (both carbachol and S. typhimurium induce Ca++ mobilization). Since basolaterally conditioned media of carbachol-stimulated epithelia contains carbachol, which, like PIF, is heat stable, such samples were applied to epithelia with and without treatment with proteinase K. Basolaterally conditioned media of carbachol-treated epithelia did not exhibit any proteinase K–sensitive induction of IL-8 secretion in virgin epithelia (Figure 3). To be certain that the residual carbachol had not simply masked a PIF response, this carbachol was removed (90%) by filtration through a 10-kDa MW cut-off filter. The retentate exhibited no more ability to induce IL-8 secretion than a similarly treated retentate (carbachol-containing) that had not been preconditioned by epithelia, indicating that carbachol, like TNF-α, does not induce PIF secretion from model epithelia.
Epithelia do not release PIF in response to TNF-α or carbachol. Model epithelia were basolaterally treated with TNF-α (20 ng/ml) or carbachol (100 μM). One hour later, basolateral media (which still contained TNF-α or carbachol) was isolated and subjected to boiling or proteinase K treatment where indicated (as described in Figure 2). Additionally, carbachol-containing media (and an equimolar solution of carbachol that had not been exposed to epithelia) were concentrated tenfold over a 10-kDa Amicon concentrator and then rediluted to the original volume. Samples were then basolaterally exposed to fresh model epithelia, and their ability to induce IL-8 secretion over a 5-hour period was measured.
PIF bioactivity can be isolated from bacterial supernatants. Having failed to obtain PIF secretion in a bacteria-free assay, we next sought to identify a PIF bioactivity in supernatants of bacteria that had not interacted with epithelial cells. The same S. typhimurium inoculum that we typically use to induce epithelial IL-8 secretion was harvested by centrifugation, washed in HBSS, and incubated for 1 hour at 37°C. The culture was then centrifuged and the supernatant filtered through a 0.2-μM filter to remove residual bacteria. This sample potently induced IL-8 secretion when applied to the basolateral surface of model intestinal epithelia (Figure 4a). Like PIF isolated from bacterial-epithelial interactions, the proinflammatory bioactivity derived from bacterial supernatant retained the ability to induce epithelial IL-8 expression when boiled, was sensitive to proteinase K, and was retained by 30 kDa cut-off filters (Figure 4b). These results indicated that PIF is likely of bacterial origin. We next investigated whether bacterial LPS was involved in this response by using polymyxin B–coated agarose to remove this compound from PIF (24). Removal of endotoxin in this manner did not result in any loss of PIF activity (control and polymyxin B bead–treated PIF induced 1.54 ± 0.23 and 1.64 ± 0.27 ng/ml IL-8 secretion, respectively). To be sure that the polymyxin B agarose did actually remove endotoxin, we verified that our treatment abolished (99.98 ± 3.2 % inhibition) the ability of a solution of LPS to activate IL-8 secretion in HUVECs (a well-characterized LPS responsive cell type). Thus, LPS is not a component of PIF.
Bacteria release a PIF with the same properties as that isolated from bacterial-epithelial interactions. An overnight culture of S. typhimurium (S.t.) was pelleted, washed two times by centrifugation, and resuspended in HBSS (1010 CFU/ml). One hour later, bacterial supernatant was isolated and basolaterally applied to model epithelia where its ability to induce IL-8 secretion was measured.(a) Supernatant was applied over a range of concentrations. (b) Bacterial supernatant was subjected to indicated treatments (as described in Figure 2) before being applied to epithelia.
PIF represents flagellin. We first sought to obtain PIF’s MW by using gel-filtration chromatography. Bacterial supernatant was filtered (0.2-μm pore size), applied to a Superose column (13-μm particle size), and fractions assayed for PIF activity by measuring their ability to induce IL-8 secretion from model epithelia. PIF bioactivity eluted as a single peak with a molecular mass of about 50 kDa. We purified PIF by an ion exchange chromatography, using as a starting material bacterial supernatants that had been boiled, filtered, and concentrated tenfold with a 30-kDa MW cut-off filter. By performing test bindings, we observed that PIF bioactivity readily bound to anion exchange, but not cation exchange resins. Thus, we used cation exchange beads to remove proteins not related to PIF bioactivity and then applied the resulting sample to a Q-Sepharose anion-exchange column. PIF bioactivity eluted from the anion-exchange column as a single peak that upon SDS-PAGE analysis contained a prominent 50-kDa band (Figure 5). This band was digested with trypsin, extracted from the gel, analyzed using amino acid sequencing (Edman degradation), and the sequences FNSAITNLG and TTSYT obtained. A search of the Salmonella database indicated that the first sequence was from FliC and the second from FljB. Thus, the band contained both of the two known S. typhimurium isoforms of the protein flagellin, the primary structural component of flagella.
Purification of PIF revealed it to be the protein flagellin. (a) Outline of PIF purification method. Numbers in parentheses correspond to lane numbers in b. (b) Coomassie-stained gel showing PIF at various stages of purification. Lane 1: MW markers (in kDa: 205, 120, 84, 52, 36, 30, 22, 7). Lane 2: S. typhimurium supernatant (i.e., starting material; too dilute to detect any bands). Lane 3: Sample concentrated tenfold over 30-kDa cut-off Amicon filter. Lane 4: Concentrated sample after incubation with S-Sepharose cation exchange beads. Lane 5: The peak bioactive anion exchange fraction. This band was sequenced and found to be flagellin. FT, flow through.
We next investigated whether we had indeed purified the protein responsible for PIF bioactivity. S. typhimurium has two highly homologous genes for flagellin, and growth conditions may dictate which isoform is expressed. Functional flagella can be made from either protein. We thus generated S. typhimurium strains deficient in flagellin (lacking the genes for both fliC and/or fljB). We isolated supernatant from these strains and their wild-type parent and measured the ability of these supernatants to induce IL-8 secretion when applied to the basolateral surface of model intestinal epithelia. In striking contrast to the wild-type strain, the flagellin-deficient mutant (fliC/fljB) supernatant did not display any IL-8–inducing bioactivity, even when added at a 1,000-fold greater concentration (Figure 6). We also analyzed whether S. typhimurium deficient in either, but not both, fliC or fljB secreted the IL-8–inducing factor. The supernatants of either of these two strains contained significant, albeit somewhat reduced, levels of IL-8–inducing activity when compared with the wild-type strain. Thus, although our wild-type cultures (originating from a single colony) contain a mix of FliC and FljB (perhaps the result of a growth-phase change during overnight incubation), either flagellin isoform can function as a PIF. The relatively reduced potencies of supernatants lacking FliC or FljB probably resulted from a lower concentration of flagellin rather than synergy between the two flagellin isoforms present in the wild-type supernatant (see below). Lastly, we verified that we could rescue the inability of flagellin-deficient S. typhimurium supernatants to induce IL-8 secretion by transforming them with plasmids encoding fliC or fljB. As shown in Figure 7b, transformation with either flagellin gene restored the ability of the organism’s supernatants to induce IL-8 secretion, further confirming our conclusion that FliC and FljB are proinflammatory factors.
Flagellin-deficient S. typhimurium do not release an IL-8 inducing bioactivity. Bacterial supernatants were isolated (as described in Figure 4) from wild-type (WT) S. typhimurium or mutant strains lacking fliC and/or fljB (“–” indicates the gene that is missing). Supernatants were then diluted as indicated, applied basolaterally to model epithelia, and IL-8 secretion assayed 5 hours later.
Various bacteria release PIF/flagellin. An overnight culture of the indicated bacterial strain was pelleted, washed two times by centrifugation, and resuspended in HBSS (1010 CFU/ml). One hour later, bacterial supernatant was isolated. Flagellin-deficient strain (fliC–/fljB–) transformed with plasmids encoding fliC and fljB is indicated by fliC–/fljB–/fliC+ and fliC–/fljB–/fljB+, respectively. (a) Supernatants were analyzed by SDS-page/immunoblotting using a mAb to flagellin. (b–d) Model epithelia were placed in HBSS. (b) Bacterial supernatants were applied basolaterally at a 1:100 dilution to model epithelia. IL-8 secretion was assayed 5 hours later. (c) Bacterial supernatants were applied basolaterally, at indicated dilution, to model epithelia. IL-8 secretion was assayed 5 hours later. (d) Live bacteria were applied to apical reservoir (108 bacteria/ epithelia). IL-8 secretion was assayed 5 hours later.
Bacterial-induced PIF responses require epithelial translocation of flagellin. Many flagellated bacteria will release flagellin into their media, and thus supernatants of most Salmonella strains would be expected to activate IL-8 secretion when applied (basolaterally, see below) to epithelia. Indeed, as shown in Figure 7, supernatants from every Salmonella strain we tested (except flagellar mutants), and five out of seven strains of nonpathogenic gut E. coli, contained flagellin as indicated by Western blotting (Figure 7a) and induced epithelial IL-8 secretion (Figure 7b) when applied basolaterally. Diluting some of these bacterial supernatants and then measuring their ability to induce IL-8 secretion indicated that the supernatants of these flagellated bacterial strains contained similar levels of PIF/flagellin bioactivity (Figure 7c). Furthermore, the PIFs isolated from these strains had the same biochemical properties as PIF/flagellin isolated from wild-type S. typhimurium (resistance to boiling, sensitivity to proteinase K; data not shown), indicating that flagellin was likely responsible for this IL-8 induction.
However, in contrast to their supernatants, the PhoPc mutant and the E. coli strains themselves elicited very little IL-8 secretion when added apically (i.e., physiologically) to model epithelia (refs. 9, 17 and Figure 7d). We reasoned that this apparent contradiction could perhaps be explained by epithelia exhibiting a polarized response to flagellin; i.e., perhaps epithelia could respond only to flagellin that reaches its basolateral surface. This was indeed the case because flagellin added apically to model epithelia failed to elicit IL-8 secretion even when added at 100-fold greater concentration than was necessary to elicit a significant response when added basolaterally (Figure 8). Therefore, as we normally add bacteria only to the apical (lumenal) surface (consistent with the physiology of the intestine), there would only be a response to flagellin if and when it is translocated across the epithelia. However, the fact that a flagellin-mediated bioactivity was isolated from the basolateral supernatants (Figure 1) indicates that indeed such translocation does occur. Additionally, Western blotting of such basolateral-conditioned media indicate that these samples do indeed contain detectable levels of flagellin within 30 minutes after apical addition of the bacteria (Figure 9a).
Basolateral flagellin, but not apical flagellin, induces IL-8 secretion. Flagellin was isolated from S. typhimurium supernatant as described in Methods, diluted as indicated, and added to the indicated reservoir of model epithelia, and IL-8 secretion was measured 5 hours later.
Flagellin rapidly appears in the basolateral supernatants of apically infected model epithelia. Model intestinal epithelia were apically exposed to indicated bacteria. Basolateral epithelial supernatants were isolated and Western blotted for flagellin. (a) Supernatants were isolated at the indicated time after addition of S. typhimurium. Isolated surface flagellin from S. typhimurium expressing FliC or FljB and PIF were directly Western blotted (i.e., not exposed to epithelia) to serve as positive controls. (b) Supernatants were isolated 1 hour after exposure to indicated organism or purified flagellin (100 ng/ml).
We next sought to gain some insight into how flagellin translocation might occur. Flagellin itself, added apically in the absence of bacteria, was unable to translocate across model epithelia (Figure 9b). Nor could flagellin translocation be mediated by a flagellin-secreting strain of normal gut E. coli. Furthermore, using our more sensitive and quantitative indicator of flagellin translocation (i.e., detecting flagellin by its IL-8–inducing bioactivity), we found that flagellin-deficient S. typhimurium could not translocate added soluble flagellin (IL-8 induction by basolateral media of epithelia apically colonized with fliC–/fljB–S. typhimurium in the presence of apical soluble flagellin was 7.3 ± 1.5 % of wild-type values). These results suggest that flagellin translocation is mediated by S. typhimurium interacting directly with flagellin and intestinal epithelia.
To begin to characterize the microbial determinants that might be required for flagellin translocation, the ability of previously characterized invasion-defective S. typhimurium mutants to mediate flagellin translocation was measured. Two regulatory mutants, PhoPc (constitutive activation of the PhoP/PhoQ regulatory system) (25) and HilΔ (spontaneous 8-kb deletion including prgH) (26), as well as the mutants invA and invG, both lacking structural components of the type III secretory apparatus (27), were used. We have shown previously that the HilΔ, but not PhoPc, mutant retains the ability to induce IL-8 secretion (7, 9). Concomitantly, HilΔ, but not PhoPc, could mediate the translocation of flagellin as measured by biodetection (Figure 10). Nonpathogenic E. coli F-18 was also unable to translocate biodetectable levels of flagellin. The S. typhimurium mutants invA and invG exhibited somewhat diminished, but not abolished, ability to translocate flagellin when compared with the wild-type strain (Figure 10). When assayed in parallel for invasion, invA and invG were found to be internalized approximately 80% and 70%, respectively, less than a wild-type strain (internalization for wild-type parent, invA, and invG was 30.7 ± 6.5, 6.0 ± 1, and 9.3 ± 4 ×104 bacteria per 0.33 cm2 epithelia). Together, these results indicate that the type III secretory-mediated interactions between S. typhimurium and epithelia may modulate, but are not absolutely required for, flagellin translocation. Moreover, whereas many flagellated Gram-negative organisms secrete flagellin, this flagellin will only activate an inflammatory response when an organism can translocate this protein across the epithelia. Thus, the ability to translocate flagellin may be an important determinant in whether a bacteria will elicit an inflammatory response.
Flagellin translocation by S. typhimurium mutants. Model epithelia were apically exposed to wild-type (WT) S. typhimurium or indicated mutant or nonpathogenic E. coli. for 45 minutes. After exposure to bacteria, basolateral supernatants were collected, boiled, filtered (through 2-μM pore size filter), and transferred to basolateral surface of virgin epithelia. IL-8 secretion was measured 5 hours later using ELISA. Wild-type represents PhoPc parent (14028s). An indistinguishable result was obtained using invA parent SR-11 (not shown).
Primary S. typhimurium induction of IL-8 occurs by a flagellin-mediated mechanism. We next sought to better assess the biological relevance of flagellin induction of epithelial IL-8 secretion. First, we measured the concentration dependence of this response. Flagellin purified from wild-type S. typhimurium (likely a mix of FliC and FljB) induced detectable IL-8 secretion at concentrations below 1 ng/ml and had an ID50 of about 2 ng/ml (Figure 11a). FljB (flagellin purified from fliC S. typhimurium) had an ID50 of 1.5 ng/ml, whereas FliC (purified from the fljB strain) had an ID50 of 2.4 ng/ml. This is more potent than IL-8 induction elicited by an equal concentration of the potent proinflammatory agonist TNF-α. In contrast, these cells do not secrete detectable levels of IL-8 in response to basolaterally added S. typhimurium LPS (no response was observed at any concentration we tested, the highest being 500 μg/ml), analogous to the results of nonpolarized epithelia (28, 29) or polarized epithelia treated apically (4, 17). Similarly, we did not observe detectable IL-8 secretion in response to two commercially available E. coli LPS preparations or LPS that we isolated from E. coli F-18. Thus, whereas LPS is thought to be important for activating subepithelial (i.e., lamina propria) macrophages, flagellin may play an important role in activating epithelial orchestration of an inflammatory response when the epithelial barrier has been breached. We next assessed whether flagellin would stimulate IL-8 production from model epithelia made from cell lines other than T84. HT29cl.19A model epithelia also secreted IL-8 in response to flagellin (unstimulated and 100 ng/ml flagellin-treated HT29cl19A epithelia secreted 0.045 ±.25 and 1.53 ± 0.45 ng/ml IL-8, respectively), indicating this response was not unique to a single cell line. Like TNF-α, and unlike bacterial toxins such as C. difficile toxin A, flagellin does not itself cause significant changes in epithelial barrier function within the time it induces IL-8 induction (Figure 11b), indicating it is likely activating an inflammatory response rather than simply being cytotoxic.
Flagellin is a potent inducer of IL-8 secretion, does not drop transepithelial electrical resistance (TER), and is essential for IL-8 secretion induced by S. typhimurium. (a) Model epithelia were basolaterally exposed to indicated concentration of LPS, flagellin, or TNF-α. IL-8 secretion was assayed 5 hours later. (b) Model epithelia were exposed (basolaterally) to HBSS (Neg), PIF (1:40 of sample used in Figure 4), TNF-α (20 ng/ml), or C. difficile toxin A (Toxin A). TER was measured as described in Methods. (c) Model epithelia were apically colonized for 45 minutes with live indicated wild-type (WT) S. typhimurium or indicated mutant strain. Nonadherent bacteria were removed by washing three times with HBSS. IL-8 secretion was measured 5 hours after initial exposure to bacteria. Flagellin-deficient strain (fliC–/fljB–) transformed with plasmids encoding fliC and fljB is indicated by fliC–/fljB–/fliC+ and fliC–/fljB–/fljB+, respectively.
Lastly, we investigated what portion of the IL-8 secretory response induced by S. typhimurium, applied in a physiologically polarized manner (i.e., apically), was accounted for by flagellin. Specifically, we examined the ability of the fliC/fljB mutant, as well as an flhD mutant (flagellar master operon), to induce IL-8 secretion when applied apically to model epithelia. Similar to their supernatants (Figure 6 and 7), neither of these live organisms elicited significant levels of IL-8 secretion (Figure 11c) from model epithelia. In contrast, strains lacking only FliC or FljB elicited IL-8 secretion to an extent that was only marginally reduced from the wild-type parent strain. Furthermore, transformation of the flagellin-deficient strain with plasmids that encode either fliC or fljB fully restored the bacteria’s ability to induce this response. Thus, flagellin is not merely an amplifier of IL-8 expression. Rather, flagellin appears to play a major role in activating epithelial orchestration of the immune inflammatory response.