Epithelial cells prime the immune response to an array of gut-derived commensals towards a tolerogenic phenotype through distinct actions of thymic stromal lymphopoietin and transforming growth factor-beta

Abstract

Humans and other mammals coexist with a diverse array of microbes colonizing the intestine, termed the microflora. The relationship is symbiotic, with the microbes benefiting from a stable environment and nutrient supply, and the host gaining competitive exclusion of pathogens and continuously maintenance of the gut immune homeostasis. Here we report novel crosstalk mechanisms between the human enterocyte cell line, Caco2, and underlying human monocyte-derived DC in a transwell model where Gram-positive (G+) commensals prevent Toll-like receptor-4 (TLR4)-dependent Escherichia coli-induced semimaturation in a TLR2-dependent fashion. These findings add to our understanding of the hypo-responsiveness of the gut epithelium towards the microflora. Gut DC posses a more tolerogenic phenotype than conventional DC. Here we show that Caco2 spent medium (SM) induces tolerogenic DC with lower expression of maturation markers, interleukin (IL)-12p70, and tumour necrosis factor-alpha when matured with G+ and Gram-negative (G-) commensals, while IL-10 production is enhanced in DC upon encountering G+ commensals and reduced upon encountering G- bacteria. The Caco2 SM-induced tolerogenic phenotype is also seen in DC priming of naive T cells with elevated levels of transforming growth factor-beta (TGF-beta) and markedly reduced levels of bacteria-induced interferon-gamma production. Caco2 cell production of IL-8, thymic stromal lymphopoietin (TSLP) and TGF-beta increases upon microbial stimulation in a strain dependent manner. TSLP and TGF-beta co-operate in inducing the tolerogenic DC phenotype but other mediators might be involved.

Figure 1

Non-polarized Caco2 cells produce IL-8, TGF-β1 and TSLP in response to G⁺ and G^– commensals. The culture supernatant of Caco2 cells was analysed for IL-8, TGF-β1 and TSLP after 18 hr stimulation with UV-killed or live commensal bacteria. The dotted line represents cytokine production in non-stimulated Caco2 cells. Live bacteria were washed and diluted to 0·01 OD units corresponding to approximately 10 µg/ml for G^– bacteria and 30 µg/ml for G⁺ bacteria. Data are means and SD of triplicate cultures. Data are representative of four experiments. Data were analysed by two-way anova and Bonferroni post-test to compare genera. Significant difference between genera is represented as *P < 0·01, **P < 0·05 and ***P < 0·001.

Figure 2

Only G^– bacteria induce semimaturation of DC underlying a Caco2 cell layer in a TLR4-dependent manner. Bacteria were added apically to fully differentiated Caco2 cells in a transwell coculture system with monocyte-derived DC present basolaterally and incubated for 24 hr. (a) Expression of surface markers on DC after apical stimuli of Caco2 cells. Filled histograms represent isotype control antibodies in the upper panel and non-stimulated DC in the lower panel, solid lines represent E. coli Nissle-stimulated Caco2 and dotted lines represent L. reuteri 12446-stimulated Caco2 in the upper panel and E. coli Nissle-stimulated Caco2 after neutralization with αTLR4 antibody apically in the lower panel. Numbers in grey represent MFI of the grey histogram, black numbers the black line and numbers in italic refers to the dotted line. (b) Basolateral TNF-α production by DC after apical stimulation of Caco2 cells as indicated with 100 µg/ml bacteria. ‘#’ indicates: not detected. Data are means and SD of duplicate cultures. Data are representative of four experiments with cells from different donors.

Figure 3

LAB abolish E. coli Nissle-induced semimaturation of underlying DC through a Caco2 cell layer in a TLR2-dependent manner. Bacteria were added apically to fully differentiated Caco2 cells in a transwell coculture system with monocyte-derived DC present basolaterally and incubated for 24 hr. (a) Expression of surface markers on DC after apical stimulation of Caco2 cells. Filled grey histograms represent no stimuli (numbers in grey refer to MFI), solid lines represent E. coli Nissle-stimulated Caco2 cells (numbers in black refer to MFI), dotted lines represent L. reuteri 12246- and E. coli Nissle-stimulated Caco2 cells (numbers in italic refer to MFI), and silver filled histograms represent stimulation with L. reuteri 12246 and E. coli Nissle after apical neutralization of Caco2 cells with anti-TLR2 antibody (numbers in silver refer to MFI). (b) Basolateral TNF-α production by DC after apical stimulation of Caco2 cells with 100 µg/ml bacteria as indicated. Data are means and SD of duplicate cultures. Data are representative of four experiments with cells from different donors.

Figure 4

The maturation pattern of human monocyte-derived DC changes in the presence of Caco2 spent medium (SM) (a) Cytokine production by DC upon maturation with commensal bacteria (5 µg/ml) in the presence or absence of Caco2 SM. Data are means and SD of triplicate cultures. Differences in DC responses in the presence or absence of Caco2 SM was tested with one-way anova with Tukey post test, ***P < 0·001, **P < 0·01 and *P < 0·05. (b) Up-regulation of costimulatory molecules and HLA-DR induced by no stimuli (iDC), B. longum Q46, L. acidophilus X37, E. coli Nissle or K. pneumonia H79129N in the presence or absence of Caco2 SM. The filled histogram is the isotype control antibody, while the solid line represents expression on DC without Caco2 SM (upper numbers refer to MFI) and the dotted line with Caco2 SM (lower numbers refers to MFI). Data are representative of four experiments with cells from different donors.

Figure 5

Caco2 spent medium (SM) changes DC priming of naïve T cells away from a Th1 response. IFN-γ and TGF-β1 produced by naïve CD4⁺ allogeneic T cells incubated for 6 days with immature DC or matured by L. acidophilus X37, L. reuteri 12246, or E. coli Nissle (1 µg/ml) in the presence or absence of Caco2 SM from three donors. CD4⁺ T cells incubated without DC did not produce measurable amounts of cytokine either in the absence or presence of Caco2 SM. Data are means and SD of triplicate cultures. T-cell cytokine responses were significantly different in the absence and presence of Caco2 SM (P < 0·01) for all donors and for all bacteria tested (except IFN-γ production induced by immature DC). Data are representative of two experiments with DC from six different donors in total.

Figure 6

The Th1 inhibiting effect of Caco2 spent medium (SM) is both TSLP and TGF-β dependent. Cytokine production by DC after maturation with L. acidophilus X37, L. reuteri 12246, E. coli Nissle (5 µg/ml) in the presence or absence of: Caco2 SM, human recombinant TSLP (100 pg/ml) and TGF-β1 (1000 pg/ml) and neutralizing αTSLP (1 µg/ml) or αTGF-β1 (2·5 µg/ml) as indicated. Data are means and SD of triplicate cultures. In the left panel, responses of differentially treated DC were tested against DC responses in the absence of Caco2 SM (black bars) with one-way anova and Tukey post test, while they in the right panel were tested against DC responses in the presence of Caco2 SM (white bars), ***P < 0·001 **P < 0·01 and *P < 0·05. This is one representative experiment out of four with cells from different donors.

Figure 7

Expression of TGF-βRI, II, III and TSLP-R is independent of the presence of Caco2 spent medium (SM). (a) Expression of TGF-βRI,II,III is independent of the presence of Caco2 SM, but is decreased by addition of rhTGF-β. (b) The expression of TSLP-R is independent of the presence of Caco2 SM, but is enhanced upon bacteria-induced maturation (5 µg/ml of L. acidophilus X37, L. reuteri 12246 or E. coli Nissle). The filled histograms represent an isotype-control antibody, the black lines represent expression in normal culture medium (upper number represents MFI), while the dotted lines represent the expression after treatment of DC as indicated (lower number represents MFI). This is one representative experiment out of three with cells from different donors.