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Bacterial DNA evokes epithelial IL-8 production by a MAPK-dependent, NF-B-independent pathway¹

Intestinal Disease Research Programme, Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada

²Correspondence: Intestinal Disease Research Programme, McMaster University, HSC-3N5C, 1200 Main St. West, Hamilton, Ontario, Canada L8N 3Z5. E-mail: mckayd{at}mcmaster.ca

SPECIFIC AIMS

Bacterial CpG DNA has been shown to activate immune cells through Toll-like receptor 9 (TLR-9). We sought to determine whether 1) enteric epithelial cell lines express TLR-9 and 2) exposure to Escherichia coli DNA would elicit an epithelial response as defined by mediator synthesis (i.e., IL-8, PGE₂) or altered barrier or ion transport.

PRINCIPAL FINDINGS

1. Expression of TLR-9 mRNA in human colonic epithelial cells
The human colonic HT-29, Caco-2, T84, and airway A549 epithelial cells were analyzed for the expression of TLR-9 by RT-PCR. THP-1 (human monocyte cell line) and CCD-18Co (human fibroblast cell line) cells served as positive and negative controls, respectively. All four epithelia constitutively expressed TLR-9 mRNA. Purification and sequencing of the PCR product from Caco-2 cells revealed a 100% match with the published human TLR-9 cDNA sequence. Demonstration of TLR-9 expression complements recent reports of other TLRs (e.g., TLR-4) on gut epithelia.

2. E. coli DNA induces epithelial IL-8 mRNA and protein
Six hours after exposure to E. coli DNA (25 µg/mL), the treated epithelia (i.e., HT-29 and Caco-2) showed up to a threefold increase in IL-8 mRNA; treatment with calf thymus DNA elicited no change in IL-8 mRNA expression. IL-8 was significantly increased 24 h post-treatment with E. coli DNA and was similarly increased when HT-29 cells were treated with CpG-rich, single-stranded oligonucleotides (Fig. 1A ). E. coli DNA evoked epithelial IL-8 synthesis in a time- and dose-dependent manner (Fig. 1B, C ). Analysis of the E. coli DNA revealed <1.7pg LPS/µg DNA and <6 ng protein/µg DNA. Treatment of the preparation with DNase obliterated its ability to evoke IL-8 production (Fig. 1A ). These controls show that the epithelial IL-8 response was due to the E. coli DNA and not a contaminant. Exposure to higher doses of LPS + E. coli DNA resulted in a significantly enhanced HT-29 cell IL-8 production.

View larger version (14K): [in this window] [in a new window]   Figure 1. Exposure to E. coli DNA results in increased epithelial IL-8 protein production. A) HT-29 cells show increased IL-8 production after a 24 h exposure to E. coli DNA (EC: 25 µg/mL) or E. coli-based synthetic oligonucleotide sequences (Oligo: 10 µg/mL), but not calf thymus DNA (CT: 25 µg/mL). IL-8 production in response to E. coli DNA was ablated by DNase treatment (n=6). B and C) Time- and dose (24 h treatment) dependency of E. coli DNA (25 µg/mL) induction of IL-8 (n=4). D) The effect of E. coli DNA (EC: 25 µg/mL) ± LPS (10 ng/mL) on epithelial IL-8 production (n=4) (*P<0.05 compared with control; **P<0.01 compared with control and other groups; mean±SE).

3. E. coli DNA evoked epithelial IL-8 production is MAPK dependent and NF-B independent
In immune cells, TLR-9 activity is dependent on endosomal acidification and hence the postulate that it is an intracellular receptor. In those studies, chloroquine was used to block endosome acidification. We found that treatment with chloroquine (10 µg/mL), though having no effect on constitutive IL-8 production, blocked the E. coli DNA-evoked IL-8 synthesis by HT-29 cells. Using a pharmacological approach, a 30 min pretreatment of epithelia with inhibitors of the ERK 1/2 (i.e., PD98059 or UO126), the p38 MAPK (i.e., SB203580), or NF-B (i.e., PDTC or SN50) pathways indicated that the E. coli DNA-evoked epithelial IL-8 synthesis was dependent on MAPKs but not on NF-B signaling (Fig. 2A ). Corroborating these data, Western blot analyses revealed increased ERK phosphorylation (correlates with enzyme activation) in extracts from E. coli DNA-treated HT-29 cells (Fig. 2A , inset), whereas EMSA analysis showed no epithelial NF-B activation (in contrast to nuclear extracts from TNF- (positive control) -treated HT-29 cells) (Fig. 2B ). E. coli DNA induced a time-dependent AP-1 signal in the treated epithelia (Fig. 2C, D ), that was reduced by pretreatment with the ERK MAPK inhibitor UO126 (Fig. 2E ).

View larger version (51K): [in this window] [in a new window]   Figure 2. A) Bar chart showing that E. coli DNA (EC: 25 µg/mL) -induced epithelial (i.e., HT-29 cell) IL-8 production is reduced or prevented by pretreatment with either inhibitor of the ERK 1/2 MAPK pathway, UO126 (25 µM) or PD98059 (50 µM), but not PDTC (100 µM) or SN50 (10 µM), which block NF-B signaling. Inset: E. coli DNA increased HT-29 phospho-ERK levels (representative of 3 experiments; equal protein loading verified by reprobing the gel for total ERK; data not shown) (n=4; *P<0.05 compared with all other groups; **P<0.01 compared with control and other groups; ***P<0.01 compared with control and other groups mean±SE). B) EMSA showing that E. coli DNA exposure did not mobilize an epithelial NF-B signal (representative of 3 experiments); NS, nonspecific band. Extracts of TNF--treated cells are included as positive control. C) EMSA showing epithelial AP-1 activation in response to E. coli DNA (EC: 25 µg/mL). Specificity of the signal is confirmed by ablation with 100-fold excess of the cold competitor (CC) and supershifting of the putative AP-1 band with an anti-pan-Fos antibody (representative of 3 experiments). D, E) Time-dependent nature of the E. coli DNA induction of AP-1 (NS, nonspecific band), and inhibition of AP-1 activation by pretreatment with the ERK MAPK inhibitor UO126 (representative of 3 experiments).

4. Exposure to E. coli DNA increases COX-2 mRNA expression but not PGE₂ production
In comparison with the IL-8 mRNA, exposure to E. coli DNA, but not calf thymus DNA, evoked a significant increase in epithelial (HT-29 and Caco-2) and THP-1 monocyte COX-2 mRNA expression (RT-PCR analysis). This increase in COX-2 mRNA did not translate into increased epithelial production of PGE₂. In contrast, E. coli DNA-treated THP-1 cells displayed a significant increase in PGE₂: THP-1 only = 1.05±0.22 vs. THP-1+E. coli DNA (25 µg/mL) = 3.49±0.51* ng/mL (*P<0.05; n=3).

5. Exposure to E. coli DNA does not affect epithelia barrier or ion transport
HT-29 and T84 epithelial cells were grown to confluence on filter supports and treated with E. coli DNA for up to 48 h. Assessment in Ussing chambers revealed no perturbations in baseline, forskolin, or carbachol-stimulated active ion transport as indicated by changes in short-circuit current. Similarly, exposure to E. coli DNA did not affect epithelial permeability as assessed by transepithelial resistance.

CONCLUSIONS AND SIGNIFICANCE

The mammalian immune system has developed under selective pressure imposed by microbial agents, and a family of receptors—the Toll-like receptors—has evolved that permit recognition of conserved, repetitive microbial elements (e.g., LPS, CpG DNA). There has been a resurgence of interest in bacteria in disease conditions ranging from inflammation to myocardial infarction. This is particularly pertinent in the context of intestinal disease since the colon is home to a microbiota in which the number of organisms is estimated to be 10-fold greater than the number of host nucleated cells. The immunostimulatory nature of many bacterial products (e.g., LPS, cell wall proteoglycans, flagellin) is well known, and evidence is emerging that bacterial DNA can be added to the list. The current study designed to test the hypothesis that bacterial DNA can directly affect epithelial function has resulted in four key findings.

First, three model human enteric epithelia were found to constitutively express TLR-9 mRNA and thus, by inference, to have the ability to respond to CpG-rich bacterial DNA. This is the first demonstration of TLR-9 expression in nonimmune cells, and supports a sentinel role for the gut epithelium in monitoring activity in the gut lumen.

Second, exposure to E. coli DNA resulted in an increase in epithelial IL-8 mRNA expression and a time- and dose-dependent increase in IL-8 synthesis. Chloroquine, an agent known to block endosomal acidification, significantly reduced E. coli DNA-induced IL-8 production. Similar findings have been presented for monocytes, suggesting commonality in the epithelial and monocyte ability to recognize and respond to bacterial DNA.

Third, pharmacological studies implicated ERK 1/2 and p38 MAPKs in the signaling pathway that converts E. coli DNA exposure into IL-8 secretion from the enterocyte. EMSA analysis revealed that the treated cells mobilized the transcription factor AP-1, which has been shown to regulate IL-8 production. Our findings have correlated well with 1) the known effects of bacterial DNA on other cell types and 2) known intracellular mechanisms that govern IL-8 production. However, numerous studies have shown that bacterial DNA elicits an NF-B response, and certainly IL-8 is an NF-B-responsive gene. We are unable to provide any data to support NF-B involvement in the E. coli DNA-induced epithelial IL-8 response: accepted pharmacological inhibitors of NF-B signaling were ineffective and EMSA analysis of nuclear extracts revealed no NF-B DNA binding activity. Thus, there would appear to be a divergence in the intracellular signals mobilized in gut epithelial (i.e., HT-29 cells) vs. immune cells in response to bacterial DNA. Although the data are surprising, it is not unprecedented. Recently, Helicobacter pylori mutants were found to mobilize an NF-B response in a monocytic but not a gastric epithelial cell line.

Fourth, exposure to E. coli DNA evoked an increase in epithelial COX-2 mRNA that was qualitatively similar to the IL-8 mRNA response. However, unlike THP-1 monocytes, the increase in COX-2 mRNA expression was not converted into increased PGE₂ synthesis by the epithelium (the production of other prostaglandins remains a possibility). Furthermore, epithelial monolayers exposed to E. coli DNA displayed no alterations in their ion transport or barrier properties: both functions are elements of the innate immune response.

We suggest that in response to bacterial DNA/TLR-9 signaling, gut epithelia increase IL-8 production via a MAPK-dependent pathway to recruit neutrophils to destroy the potential bacterial intruder while maintaining an intact epithelial barrier. Simultaneously, other genes are up-regulated but held in check, possibly until additional proinflammatory signals are received (e.g., LPS or TNF-) (Fig. 3 ). This initially protective response, if prolonged, has the potential to evoke or exaggerate inflammatory disease at mucosal surfaces such as the gut, airway, and urogenital tract.

View larger version (24K): [in this window] [in a new window]   Figure 3. Schematic diagram illustrating the interaction of bacterial DNA with epithelium.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0950fje; doi: 10.1096/fj.02-0950fje

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This Article Full Text (PDF) All Versions of this Article: 17/10/1319 02-0950fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by AKHTAR, M. Articles by MCKAY, D. M. Search for Related Content PubMed PubMed Citation Articles by AKHTAR, M. Articles by MCKAY, D. M.