Influenza A virus infection of intestinal epithelial cells enhances the adhesion ability of Crohn's disease associated Escherichia coli strains

Abstract

Modifications of intestinal glycoreceptors expression, in particular CEACAM6, typically found in ileal Crohn's disease (CD), favor, among the commensal species of microbiota, the enrichment in Escherichia coli. Removal of protein glycosidic residues by neuraminidase, a sialidase typical of influenza virus, increases adhesion ability of Escherichia coli to Caco-2 intestinal cells. In this study we investigated whether influenza virus infection of human intestinal epithelial cells could influence the adhesiveness of different Escherichia coli strains isolated from CD patients by altering surface glycoreceptors. Influenza virus infection of intestinal cells increased exposure of galactose and mannose residues on the cell surface. In particular, glycoreceptors Thomsen-Friedenreich and CEACAM6 were over-expressed in influenza virus infected cells. In the same experimental conditions, a significant increase in bacterial adhesiveness was observed, independently of their own adhesive ability. The increase was reverted by treatment with anti-TF and anti-CEACAM6 antibodies. Interestingly, influenza virus was able to efficiently replicate in human primary intestinal cells leading to TF exposure. Finally, intestinal infected cells produced high levels of pro-inflammatory cytokines compared to control. Overall these data suggest that influenza virus infection, could constitute an additional risk factor in CD patients.

Fig 1. Influenza virus increases exposure of galactose residues in intestinal cells.

Caco-2 cells were infected with PR8 at 0.2 and 0.8 MOI and maintained for 4, 6 and 24 hrs at 37°C. Cells were then fixed and samples were incubated with anti-HA Abs (upper panel) to follow the progression of viral infection and with PNA (bottom panel) to detect galactose residues in PR8 infected cell and in the control cell (Mock-I), as described in Methods. BF: Bright field. Results are shown for one representative experiment of the three performed at magnification of 100X.

Fig 2. Influenza virus increases exposure of mannose residues in intestinal cells.

Caco-2 cells were infected with PR8 at 0.2 and 0.8 MOI and maintained for 4, 6 and 24 hrs at 37°C. Cells were then fixed and samples were incubated with anti-HA Abs (upper panel) to follow the progression of viral infection and with ConA (bottom panel) to detect mannose residues, in PR8 infected cell and in the control cell (Mock-I), as described in Methods. BF: Bright field. Results are shown for one representative experiment of the three performed at magnification of 100X.

Fig 3. Influenza virus enhances exposure of TF antigen and cytokines secretion in intestinal cells.

A) Caco-2 cells were infected with PR8 at 0.2 and 0.8 MOI and maintained for 4, 6 and 24 hrs at 37°C. Cells were fixed and incubated with anti-TF Ab in PR8 infected cells and in the control cells (Mock-I) as described in Methods. Results are shown for one representative experiment of the three performed at magnification of 100X. B) Viral replication was assessed by haemagglutination (HAU/ml) assays in the supernatant of infected cells. C) Cells were infected at 0.8 MOI and supernatants were collected at 24 and 48 hrs p.i. The concentration of pro-inflammatory cytokines (TNF-α, IL-6 and RANTES) was measured by Bioplex multiplex assay. Data are expressed as fold induction of pro-inflammatory cytokines in infected cells relative to control cells. The graphs represent cumulative results of two different experiments.

Fig 4. Influenza virus enhances exposure of CEACAM6 receptor in intestinal cells.

Caco-2 cells were infected with PR8 at 0.2 and 0.8 MOI and maintained for 4, 6 and 24 hrs at 37°C. Cells were then fixed and samples were incubated with anti-HA to assess viral infection or anti-CEACAM6 Abs to detect antigen expression in PR8 infected cell and in the control cell (Mock-I), (see Methods). BF: Bright field. Results are shown for one representative experiment of the three performed at magnification of 100X.

Fig 5. Influenza virus leads to enhanced CEACAM6 mRNA level and protein expression.

Caco-2 cells were infected with PR8 (0.8 MOI) and harvested at different times p.i. Samples were used for mRNA or for protein extraction, as described in Methods. A: real-time PCR of CEACAM6 mRNA levels in infected cells harvested at 4, 6, 18 and 24 hrs p.i. Levels of mRNA are expressed as the ratio between CEACAM6 normalized for β-actin and GAPDH. B left panel: a rapresentative western blot analysis of CEACAM6 expression at different times 4, 6 and 24 hrs p.i. As controls, mock-infected cells were recovered at 4 and 24 hrs p.i. Samples were lysed, run onto SDS-PAGE, blotted and immunostained with anti-CEACAM6 Abs. Loading control was Actin. B right panel: densitometric analysis expressed as the ratio of CEACAM6 normalized for actin is reported. The values of western blot signals were obtained by densitometric analysis of control at 4 and 24 hrs respect to infected cell at 4, 6 and 24. Results are shown for the three representative experiments (*P<0.05, **P<0.01).

Fig 6. Anti-TF and anti-CEACAM6 Abs reduce bacterial adhesion on PR8-infected cells.

Caco-2 cells were infected with PR8 (0.8 MOI) for 24 hrs p.i., and then were incubated with anti-TF or anti-CEACAM6 Abs. After 2 hrs incubation, cells were infected with E. coli S15 (A), LF82 (B) and LF82-ΔfimH mutant (C) strains, as described in Methods. Data represent the mean ± SD of three independent experiments, each performed in triplicate *P<0.05 vs. untreated infected cells.

Fig 7. Influenza virus enhances exposure of TF antigen and cytokines secretion in primary cells.

A) Viral replication was assessed in the supernatants of intestinal primary infected cells by real time RT-PCR and expressed as number of viral M1 RNA copies/ml. B) Primary intestinal cells were infected with PR8 at 1.6 MOI at 33°C and supernatants were collected at 24 hrs p.i. The concentration of pro-inflammatory cytokines (TNF-α and IL-6) was measured by Bioplex multiplex assay. Data are expressed as fold induction of pro-inflammatory cytokines in infected cells relative to control cells. The graphs represent cumulative results of two different experiments C) Primary were infected with PR8 at 1.6 MOI for 24 hrs at 33°C. Mock infected (MOCK-I) and infected cells were fixed and incubated with anti-TF Ab as described in Methods. Results are shown for one representative experiment of the two performed at magnification of 100X.