James L. Watson

Bacterial DNA evokes epithelial IL-8 production by a MAPK-dependent, NF-κB-independent pathway

Recognition of bacterial products by the innate immune system is dependent on pattern‐recognition receptors: toll‐like receptor 9 (TLR‐9) in the case of bacterial DNA. We hypothesized that bacterial DNA can directly affect enteric epithelial cells. RT‐PCR revealed constitutive TLR‐9 mRNA expression in three human colonic epithelial cell lines (T84, HT‐29, Caco‐2) and THP‐1 monocytes. Epithelial cells, in six‐well culture plates or on filter supports, were exposed to E. coli DNA (1–50 µg/ml), synthetic CpG‐rich oligonucleotides, or calf thymus DNA for 6–48 h. Exposure to E. coli DNA resulted in an increase in IL‐8 mRNA, and a time‐and dose‐dependent increase in IL‐8 secretion. Also, CpG oligonucleotides induced epithelial IL‐8 production, whereas calf thymus DNA did not. Exposure to E. coli DNA resulted in phosphorylation of ERK 1/2 MAPK and inhibitors of ERK activity (PD98059, UO126) significantly reduced the evoked IL‐8 production. In contrast, inhibitors of NFκB activity (PDTC, SN50) did not block E. coli DNA‐induced IL‐8 production. Electrophoretic mobility shift assays revealed that E. coli DNA stimulated epithelial AP‐1 but not NFκB activation. The barrier (i.e., transepithelial resistance) and ion transport parameters of epithelial monolayers (assessed in Ussing chambers) were unaltered following E. coli DNA exposure. Thus model gut epithelia express TLR‐9 mRNA and, while maintaining their barrier function, can respond to E. coli DNA by increased IL‐8 production.

Epithelia Under Metabolic Stress Perceive Commensal Bacteria as a Threat

The normal gut flora has been implicated in the pathophysiology of inflammatory bowel disease and there is increased interest in the role that stress can play in gut disease. The chemical stressor dinitrophenol (DNP, uncouples oxidative phosphorylation) was injected into the ileum of laparotomized rats and mitochondria structure, epithelial permeability, and inflammatory cell infiltrate were examined 6 and 24 hours later. Monolayers of human colonic epithelial cells (T84, HT-29) were treated with DNP +/- commensal Escherichia coli, followed by assessment of epithelial permeability, bacterial translocation, and chemokine (ie, interleukin-8) synthesis. Delivery of DNP into rat distal ileum resulted in disruption of epithelial mitochondria; similar changes were noted in mildly inflamed ileal resections from patients with Crohns disease. Also, DNP-treated ileum displayed increased gut permeability and immune cell recruitment. Subsequent studies revealed deceased barrier function, increased bacterial translocation, increased production of interleukin-8, and enhanced mobilization of the transcription factor AP-1 in the model epithelial cell lines exposed to commensal bacteria (E. coli strains HB101 or C25), but only when the monolayers were pretreated with DNP (0.1 mmol/L). These data suggest that enteric epithelia under metabolic stress perceive a normally innocuous bacterium as threatening, resulting in loss of barrier function, increased penetration of bacteria into the mucosa, and increased chemokine synthesis. Such responses could precipitate an inflammatory episode and contribute to existing enteric inflammatory disorders.

Phosphatidylinositol 3′-Kinase Is a Critical Mediator of Interferon-γ-Induced Increases in Enteric Epithelial Permeability

The epithelial lining of mucosal surfaces acts as a barrier to regulate the entry of antigen and pathogens. Nowhere is this function of the contiguous epithelium more important than in the gut, which is continually exposed to a huge antigenic load and, in the colon, an immense commensal microbiota. We assessed the intracellular signaling events that underlie interferon (IFN) γ-induced increases in epithelial permeability using monolayers of the human colonic T84 epithelial cell line. Confluent epithelial monolayers on semipermeable supports were treated with IFNγ (20 ng/ml), and barrier function was assessed 48 h later by measuring transepithelial electrical resistance (TER: reflects passive ion flux), fluxes of 51Cr-EDTA and horseradish peroxidase (HRP), and transcytosis of noninvasive, nonpathogenic Escherichia coli (strain HB101). Exposure to IFNγ decreased barrier function as assessed by all four markers. The phosphatidylinositol 3′-kinase (PI-3K) inhibitors, LY294002 [2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride] and wortmannin, did not affect baseline permeability characteristics but completely blocked the drop in TER, increased fluxes of 51Cr-EDTA and HRP, and significantly reduced E. coli transcytosis evoked by IFNγ. In addition, use of the pan-protein kinase C (PKC) inhibitor, bisindolylmaleimide I (5 μM), but not rottlerin (blocks PKCδ), partially ameliorated the drop in TER and inhibited increased E. coli transcytosis. Addition of the PI-3K and PKC inhibitors to epithelia 6 h after IFNγ exposure still prevented the increase in paracellular permeability but not E. coli transcytosis. Thus, IFNγ-induced increases in epithelial paracellular and transcellular permeability are critically dependent on PI-3K activity, which may represent an epithelial-specific target to treat immune-mediated loss of barrier function.