Escherichia coli Nissle 1917 distinctively modulates T-cell cycling and expansion via toll-like receptor 2 signaling

Abstract

Although the probiotic Escherichia coli strain Nissle 1917 has been proven to be efficacious for the treatment of inflammatory bowel diseases, the underlying mechanisms of action still remain elusive. The aim of the present study was to analyze the effects of E. coli Nissle 1917 on cell cycling and apoptosis of peripheral blood and lamina propria T cells (PBT and LPT, respectively). Anti-CD3-stimulated PBT and LPT were treated with E. coli Nissle 1917-conditioned medium (E. coli Nissle 1917-CM) or heat-inactivated E. coli Nissle 1917. Cyclin B1, DNA content, and caspase 3 expression were measured by flow cytometry to assess cell cycle kinetics and apoptosis. Protein levels of several cell cycle and apoptosis modulators were determined by immunoblotting, and cytokine profiles were determined by cytometric bead array. E. coli Nissle 1917-CM inhibits cell cycling and expansion of peripheral blood but not mucosal T cells. Bacterial lipoproteins mimicked the effect of E. coli Nissle 1917-CM; in contrast, heat-inactivated E. coli Nissle 1917, lipopolysaccharide, or CpG DNA did not alter PBT cell cycling. E. coli Nissle 1917-CM decreased cyclin D2, B1, and retinoblastoma protein expression, contributing to the reduction of T-cell proliferation. E. coli Nissle 1917 significantly inhibited the expression of interleukin-2 (IL-2), tumor necrosis factor alpha, and gamma interferon but increased IL-10 production in PBT. Using Toll-like receptor 2 (TLR-2) knockout mice, we further demonstrate that the inhibition of PBT proliferation by E. coli Nissle 1917-CM is TLR-2 dependent. The differential reaction of circulating and tissue-bound T cells towards E. coli Nissle 1917 may explain the beneficial effect of E. coli Nissle 1917 in intestinal inflammation. E. coli Nissle 1917 may downregulate the expansion of newly recruited T cells into the mucosa and limit intestinal inflammation, while already activated tissue-bound T cells may eliminate deleterious antigens in order to maintain immunological homeostasis.

FIG. 1.

E. coli Nissle 1917-CM inhibits cell cycle progression of PBT. (A) E. coli Nissle 1917-CM dose dependently decreases the number of CD3-activated PBT in the S and G₂/M phase. (B) CM (50% [vol/vol]) from the E. coli strains DSM 498, PZ 840, PZ 873, and PZ 915 decrease the number of CD3-activated PBT in the S and G₂/M phase. Freshly isolated PBT were cultured without stimulation (unstim) or in the presence of cross-bound CD3 MAbs (stim) and in the presence of 0, 10, 25 and 50% (vol/vol) E. coli Nissle 1917-CM stimulated for 3 days as described in Materials and Methods. Each phase of the cell cycle was assessed by measuring DNA content by PI staining followed by flow cytometry. The graphs in panel A are representative of seven different experiments, and the graphs in panel B are representative of three different experiments. EcN, E. coli Nissle 1917.

FIG. 2.

Differential expression of cell cycle regulators in PBT following culture with E. coli Nissle 1917-CM. Immunoblot analysis shows downregulated expression of cyclin D2, Rb protein phosphorylation (pRb), E2F-1, and cyclin A following culture with E. coli Nissle 1917-CM as outlined in Materials and Methods. In contrast, the expression of cell cycle inhibitors p21 and p53 is not altered by E. coli Nissle 1917-CM. Freshly isolated PBT were cultured with anti-CD3 MAbs in the presence of 0, 10 and 25% (vol/vol) E. coli Nissle 1917-CM for 3 days, after which expression of cell cycle regulators was assessed by Western blotting. Each panel is representative of four different experiments. EcN, E. coli Nissle 1917.

FIG. 3.

Culture with E. coli Nissle 1917-CM reduces the expression of cyclin B1 in PBT. Flow cytometric analysis shows decreased cyclin B1 expression in the G₂/M phase of CD3-activated PBT compared to control cells following culture with E. coli Nissle 1917-CM. Freshly isolated PBT were cultured with anti-CD3 MAbs in the presence of 0, 10, 25 and 50% (vol/vol) E. coli Nissle 1917-CM for 3 days, after which cyclin B1 expression and DNA content were examined by flow cytometry. The figure is representative of seven different experiments. EcN, E. coli Nissle 1917.

FIG. 4.

E. coli Nissle 1917-CM does not induce T-cell apoptosis or necrosis in PBT. Analysis of annexin V and PI levels revealed that E. coli Nissle 1917-CM does not induce cell death in anti-CD3-activated PBT. Freshly isolated PBT were cultured with anti-CD3 MAb in the presence of 0, 10, 25 and 50% (vol/vol) E. coli Nissle 1917-CM for 2 days, after which apoptosis and necrosis were assessed by annexin V and PI staining, respectively. The figure is representative of three different experiments showing similar results. EcN, E. coli Nissle 1917.

FIG. 5.

Expansion of CD3-activated PBT in the presence of E. coli Nissle 1917-CM. CD3 activation induces the generation of at least four cell divisions comprising 48% of the original population in PBT. In contrast, when 10% (vol/vol) E. coli Nissle 1917-CM was added, only 23% of similarly activated PBT expanded and generated at most three daughter cell populations. The addition of 25 or 50% E. coli Nissle 1917-CM nearly completely eliminated PBT expansion. The first peak to the right represents the undivided cell population. The numbers indicate the percentages of divided cells distributed in the subsequent peaks. Freshly isolated PBT were incubated with 5 μM CFDA SE and then cultured with anti-CD3 and anti-CD28 MAbs and IL-2 in the presence of 0, 10, 25 and 50% (vol/vol) E. coli Nissle 1917-CM for 4 days, and cell divisions were determined by flow cytometry. The figure is representative of four different experiments. EcN, E. coli Nissle 1917.

FIG. 6.

E. coli Nissle 1917-CM does not inhibit cell cycle progression of LPT. The number of anti-CD3- or anti-CD2-stimulated LPT in the S and G₂/M phase remains unchanged, regardless whether 0, 10, 25 or 50% (vol/vol) E. coli Nissle 1917 was added. Freshly isolated LPT were stimulated for 3 days with anti-CD3 or anti-CD2 MAbs and cultured in the presence of 0, 10, 25 and 50% (vol/vol) E. coli Nissle 1917-CM. Each phase of the cell cycle was assessed by measuring DNA content by PI staining followed by flow cytometry. The graphs are representative of four different experiments. EcN, E. coli Nissle 1917.

FIG. 7.

Expansion of LPTs in the presence of E. coli Nissle 1917-CM. CD3- and CD2-activated LPT generated one daughter cell population, comprising about 30% of the cell population. LPT expansion remained unchanged in the presence of 10, 25 and 50% (vol/vol) E. coli Nissle 1917-CM. The first peak to the right represents the undivided cell population. The numbers indicate the percentages of divided cells distributed in the subsequent peaks. Freshly isolated LPT were incubated with 5 μM CFDA SE and then cultured with anti-CD3 and anti-CD28 MAbs and IL-2 or with anti-CD2 and anti-CD28 MAbs and IL-2 in the presence of 0, 10, 25 and 50% (vol/vol) E. coli Nissle 1917-CM for 4 days, and cell divisions were determined by flow cytometry. The figure is representative of four different experiments. EcN, E. coli Nissle 1917.

FIG. 8.

E. coli Nissle 1917-CM reduces cell cycling of CD45RA⁺ PBT and CD45RO⁺ PBT. The number of CD3-activated PBT in the S and G₂/M phase at 3 days dropped comparably in CD45RA⁺ and CD45RO⁺ PBT. Freshly isolated PBT were sorted in CD45RA⁺ PBT and CD45RO⁺ PBT and cultured with anti-CD3 MAbs in the presence of 0 and 25% (vol/vol) E. coli Nissle 1917-CM for 3 days, after which cyclin B1 expression and DNA content were examined by flow cytometry. The figure is representative of three different experiments. EcN, E. coli Nissle 1917.

FIG. 9.

Distinct cytokine expression profiles induced by E. coli Nissle 1917-CM in PBT. E. coli Nissle 1917-CM dose dependently and significantly inhibited IL-2, TNF-α, and IFN-γ production in PBT, while IL-10 production was markedly upregulated. × Freshly isolated PBT (10⁵ cells) were cultured for 3 days without stimulation or in the presence of anti-CD3 MAb and 0, 10, and 25% (vol/vol) E. coli Nissle 1917-CM. Supernatants were then collected, and cytokine secretion was analyzed by using a cytometric bead array assay. Each bar represents mean ± SEM of three experiments. *, P < 0.05 for change versus CD3 with 0% E. coli Nissle 1917-CM. EcN, E. coli Nissle 1917.

FIG. 10.

Effect of pathogen-associated molecular patterns on PBT cell cycle progression. E. coli Nissle 1917-CM inhibits PBT cell cycling of anti-CD3-activated PBT, regardless whether the CM is frozen at −80°C or heat inactivated as outlined in Materials and Methods. When E. coli Nissle 1917 was heat inactivated before the T-cell medium was added, cell cycling was not impaired. Whereas E. coli Nissle 1917 LPS and CpG DNA did not modulate PBT cycling, BLPs mimicked the effect of E. coli Nissle 1917-CM and substantially decreased PBT cycling. Freshly isolated PBT were stimulated for 3 days with anti-CD3 MAb and cultured in the presence of the respective substances as described in Materials and Methods. The percentages of cells in the S/G₂/M phase was assessed by measuring DNA content by PI staining followed by flow cytometry. Each bar represents the mean ± SEM of three to four experiments. *, P < 0.05 for change versus control (0% E. coli Nissle 1917-CM). EcN, E. coli Nissle 1917.

FIG. 11.

E. coli Nissle 1917-CM does not inhibit cell cycle progression in TLR-2 deficient mice. E. coli Nissle 1917-CM dose dependently decreases the number of CD3-activated PBT in the S and G₂/M phase in wild-type mice but not TLR-2^−/− littermates. Freshly isolated PBT were stimulated for 3 days with mouse anti-CD3 MAb and cultured in the presence of 0, 10, and 25% (vol/vol) E. coli-CM and BLPs (100 ng/ml). Each phase of the cell cycle was assessed by measuring DNA content by PI staining followed by flow cytometry. The graphs are representative of two different experiments. EcN, E. coli Nissle 1917.