Hypoxia-inducible factor-dependent regulation of platelet-activating factor receptor as a route for gram-positive bacterial translocation across epithelia

Abstract

Mucosal surfaces, such as the lung and intestine, are lined by a monolayer of epithelia that provides tissue barrier and transport function. It is recently appreciated that a common feature of inflammatory processes within the mucosa is hypoxia (so-called inflammatory hypoxia). Given the strong association between bacterial translocation and mucosal inflammatory disease, we hypothesized that intestinal epithelial hypoxia influences bacterial translocation. Initial studies revealed that exposure of cultured intestinal epithelia to hypoxia (pO(2), 20 torr; 24-48 h) resulted in a increase of up to 40-fold in the translocation of some strains of Gram-positive bacteria, independently of epithelial barrier function. A screen of relevant pathway inhibitors identified a prominent role for the platelet-activating factor receptor (PAFr) in hypoxia-associated bacterial translocation, wherein pharmacologic antagonists of PAFr blocked bacterial translocation by as much as 80 +/- 6%. Extensions of these studies revealed that hypoxia prominently induces PAFr through a hypoxia-inducible factor (HIF)-dependent mechanism. Indeed, HIF and PAFr loss of function studies (short hairpin RNA) revealed that apically expressed PAFr is central to the induction of translocation for the Gram-positive bacteria Enterococcus faecalis. Together, these findings reveal that some strains of Gram-positive bacteria exploit HIF-regulated PAFr as a means for translocation through intestinal epithelial cells.

Figure 1.

Increased translocation of E. faecalis across hypoxic Caco-2 monolayers. (A) Influence of hypoxia on the translocation of a selection of Gram-negative and Gram-positive over 60 min. (B) Influence of hypoxia on the translocation of E. faecalis over time. (C) The rate of increased E. faecalis translocation with hypoxia. (D) Apparent permeability of Caco-2 monolayers after infection, TNF-α (10 ng/ml) is a positive control. (E) Translocation of E. faecalis across Caco-2 monolayers is temperature dependent. Results are derived from six or more experiments. Hx, hypoxia; Nx, normoxia, *p < 0.05, **p < 0.01, and ***p < 0.005 (Student's t test).

Figure 2.

PAFr antagonist CV-6209 (60 μM) prevents E. faecalis but not S. mitis or E. coli translocation across hypoxic Caco-2 monolayers. (A) Influence of CV-6209, a PAFr antagonist, and C-PAF, a PAFr agonist, on the translocation of E. faecalis across Caco-2 monolayers. (B) Influence of PAFr antagonism on the translocation of S. mitis across Caco-2 monolayers. (C) Influence of PAFr antagonism on the translocation of E. coli across Caco-2 monolayers. (D) Inhibition E. faecalis translocation by CV-6209 over time. Bacterial translocation over 60 min. Results are derived from six or more experiments. Hx, hypoxia; Nx, normoxia. **p < 0.01 and ***p < 0.005 (Student's t test).

Figure 3.

Induction of PAFr in hypoxic Caco-2 monolayers and in DSS colitis. (A) Induction of PAFr mRNA transcript over time in Caco-2 monolayers incubated in hypoxia. (B) Expression of PAFr protein in Caco-2 monolayers after 24 h in hypoxia; 48 kDa represents apical cell surface PAFr (verified by biotinylation, densitometry analysis 3191 ± 248 pixels for apical PAFr vs. 319 ± 254 pixels for basolateral PAFr), whereas the 69-kDA ban represents cytoplasmic PAFr. (C) Densitometric analysis of PAFr protein expression by ImageJ pixel count. Results are derived from three experiments. (D) Apical (ap) and basolateral (bas) expression of PAFr in hypoxic and normoxic Caco-2 monolayers. (E) Induction of PAFr in DSS colitis. mRNA was isolated from epithelial cells from mice 6 d after induction of DSS colitis. (F) PAFr correlation with disease severity (weight loss). Results are derived from three experiments. For A, *p < 0.05 by one-way ANOVA. For B, p < 0.05 by linear regression analysis. Hx, hypoxia; Nx, normoxia. *p < 0.05 and ***p < 0.005 (Student's t test).

Figure 4.

shRNA-mediated repression of PAFr in Caco-2 monolayers reduces translocation of E. faecalis. (A) PAFr is not induced by hypoxia in PAFr knockdown Caco-2 monolayers incubated in hypoxia. (B) Loss of protein induction by PAFr knockdown Caco-2 monolayers after 24 h in hypoxia. (C) Translocation of E. faecalis across hypoxic and normoxic PAFr knockdown Caco-2 monolayers. CV-6209 (60 μM) does not reduced translocation across PAFr knockdown Caco-2 monolayers. Bacterial translocation was over 120 min. Results are derived from six experiments. Hx, hypoxia; Nx, normoxia. **p < 0.01 and ***p < 0.005 (Student's t test).

Figure 5.

E. faecalis adherence-to and invasion-of scr-shRNA and PAFr-shRNA Caco-2 monolayers. (A) Fluorescent staining of PAFr and E. faecalis on Caco-2 scr-shRNA monolayers. (B) Fluorescent staining of PAFr and E. faecalis on hypoxic Caco-2 scr-shRNA monolayers. (C) Fluorescent staining of PAFr and E. faecalis on hypoxic Caco-2 PAFr-shRNA monolayers. (D) Internalization of E. faecalis by hypoxic hypoxic Caco-2 PAFr-shRNA monolayers over time. Magnification at 40× for top-down fluorescent imaging and at 63× for confocal X-Z imaging, Blue, DAPI nuclear stain (Invitrogen); red, anti-PAFr antibody (Cayman Chemical); and green, BacLight labeled E. faecalis (Invitrogen). Results are derived from three experiments and images are representative.

Figure 6.

Knockdown of HIF-1 in Caco-2 monolayers prevents PAFr induction in hypoxia and the translocation of E. faecalis. (A) Expression of both HIF-1α and HIF-2α isoforms in intestinal epithelial cells. Western blot analysis of HIF knockdown Caco-2 cells cultured for 16 h under normoxic conditions (Nx) or in hypoxia (Hx) with the prolyl hydroxylase domain inhibitor dimethyloxallyl glycine (DMOG; 0.5 mg/ml). Nuclear lysates were immunoprobed with antibodies against HIF-1α and HIF-2α. TATA-binding protein levels were monitored as a reference loading control. Highest level of repression was observed in the stable cell line harboring shRNA 4 (70% repression); this cell line was used in all further experiments. sh, short hairpin RNA. (B) Loss of PAFr is not induced by hypoxia in HIF-1 knockdown Caco-2 monolayers incubated in hypoxia. (C) PAFr protein induction is reduced in HIF-1 knockdown Caco-2 monolayers after 24 h in hypoxia. (D) Translocation of E. faecalis across hypoxic and normoxic HIF-1 knockdown Caco-2 monolayers. (E) ChIP analysis to examine HIF-1 binding to the PAFr promoter in hypoxic Caco-2 cells. Reaction controls included immunoprecipitations using nonspecific IgG and beads only as well as by PCR performed using total Caco-2 DNA (Input). Results are derived from three to six experiments. Hx, hypoxia; Nx, normoxia. *p < 0.05, **p < 0.01, and ***p < 0.005 (Student's t test).