Human intestinal epithelial cells express interleukin-10 through Toll-like receptor 4-mediated epithelial-macrophage crosstalk

Abstract

In the intestine, interaction between epithelial cells and macrophages (MΦs) create a unique immunoregulatory microenvironment necessary to maintain local immune and tissue homeostasis. Human intestinal epithelial cells (IECs) have been shown to express interleukin (IL)-10, which keeps epithelial integrity. We have demonstrated that bacterial signaling through Toll-like receptor (TLR) 4 induces 15-deoxy-Δ-12,14-prostaglandin J2 (15d-PGJ2) synthesis in intestinal MΦs by cyclooxygenase (Cox)-2 expression. Here, we show that TLR4 signaling generates crosstalk between IECs and MΦs that enhances IL-10 expression in IECs. Direct stimulation of TLR4 leads to the expression of IL-10 in IECs, while the presence of MΦs in a Transwell system induces another peak in IL-10 expression in IECs at a later time point. The second peak of the IL-10 expression is two times greater than the first peak. This late induction of IL-10 depends on the nuclear receptor peroxisome proliferator-activated receptor (PPAR) γ that is accumulated in IECs by TLR4-mediated inhibition of the ubiquitin-proteasomal pathway. TLR4 signaling in MΦs in turn synthesizes 15d-PGJ2 through p38 and ERK activation and Cox-2 induction, which activates PPARγ in IECs. These results suggest that TLR4 signaling maintains IL-10 production in IECs by generating epithelial-MΦs crosstalk, which is an important mechanism in the maintenance of intestinal homeostasis mediated through host-bacterial interactions.

Fig. 1

Human primary IECs express IL-10. a Morphological observation of isolated IECs. Representative picture of IECs observed under the phase contrast light microscope is shown (original magnification ×40, bar = 5 μm). b RT-PCR analysis of mRNA expression of human IL-10, TLR4 and PPARγ in isolated IECs. Three independent IEC samples are shown (lanes 2–4). Lamina propria mononuclear cells (LPMCs, lane 1) were used as a control to prove no contamination with hematopoietic cells showing CD45 mRNA expression (middle panel).

Fig. 2

Presence of MΦs amplifies IL-10 production by human IECs in response to LPS. a Real-time PCR results of IL-10 mRNA expression in SW480-APC cells grown in conventional wells. Cells were stimulated with LPS for the indicated periods. The relative expression of hIL-10 to β-actin is shown. Data from three independent experiments of triplicate samples with SEM (* p < 0.05). b Real-time PCR results of IL-10 mRNA expression in polarized SW480-APC cells (IEC) in the Transwell system. Cells were basolaterally stimulated with LPS for the indicated periods. One series of polarized SW480-APC cells (inserts) were co-cultured with mouse peritoneal MΦs (bottom wells). Dotted lines show results from LPS-non-treated controls. The relative expression of hIL-10 to β-actin is shown. Data from three independent experiments of triplicate samples with SEM (* p < 0.05). c IL-10 production from polarized SW480-APC cells with or without co-culturing with mouse peritoneal MΦs in the Transwell system. Cells were basolaterally stimulated with LPS as indicated. hIL-10 concentration in the bottom wells was measured by specific ELISA. Data from three independent experiments of duplicate samples with SEM (* p < 0.05). d Real-time PCR results of IL-10 mRNA expression in polarized SW480-APC cells during co-culturing with human monocyte/MΦ line U937 cells in the Transwell system. Cells were basolaterally stimulated with LPS for the indicated periods. The relative expression of hIL-10 to β-actin is shown. Data from three independent experiments of triplicate samples with SEM (* p < 0.05). e IL-10 production from mouse peritoneal MΦs during co-culture with SW480-APC cells in the Transwell system. Cells were basolaterally stimulated with LPS as indicated. Mouse IL-10 (mIL-10) concentration in the bottom wells was measured by specific ELISA. Data from two independent experiments of duplicate samples with SEM (* p < 0.05). f ELISA detection of hIL-10 without cross-reaction to mouse IL-10. Black bars show concentration of IL-10 (human: wells A, B, C, mouse: wells F, G, H) added and white bars show optical density (OD) measured by a spectrophotometer. Wells D and E received no IL-10 (background).

Fig. 3

LPS stimulates MΦ 15d-PGJ2 synthesis through TLR4-dependent activation of p38, ERK MAP kinases and Cox-2 induction during co-culturing with IECs. a 15d-PGJ2 synthesis in IEC-MΦ co-culture. Polarized SW480-APC cells were co-cultured with mouse peritoneal MΦs in the Transwell system. LPS was added to the bottom wells. Supernatant from the bottom wells was measured for 15d-PGJ2 by EIA at the indicated periods. Data from three independent experiments of duplicate samples with SEM (* p < 0.05). b 15d-PGJ2 synthesis by cell types. Mouse peritoneal MΦs were co-cultured with or without polarized SW480-APC cells (IECs) in the Transwell system. LPS was added to the wells of MΦs. Polarized IECs without MΦs were basolaterally stimulated with LPS as indicated. Supernatant from the bottom wells was measured for 15d-PGJ2 at the indicated periods. Data from three independent experiments of duplicate samples with SEM (* p < 0.05). c 15d-PGJ2 synthesis during inhibition of Cox-2 or PI3 and MAP kinases. Polarized SW480-APC cells were co-cultured with mouse peritoneal MΦs in the Transwell system. To block TLR4 signaling, some wells were prepared with MΦs from TLR4-/- mice. Inhibitors for p38 (SB203580; 20 μM), JNK (SP600125; 20 μM), ERK (PD98059; 20 μM), PI3K (LY294002; 20 μM) and Cox-2 (NS398; 5 μM) were added to the bottom wells. Ninety minutes later, LPS was added to the bottom wells. Supernatant from the bottom wells was measured for 15d-PGJ2 after 48 h. Data from two independent experiments of triplicate samples with SEM (* p < 0.05). d Western blot analysis of Cox-2 in MΦs. Mouse peritoneal MΦs were co-cultured with SW480-APC cells in the Transwell system. Protein samples were harvested from MΦs before and after 24 h of LPS stimulation in the bottom wells. An equal amount of protein was loaded per lane. The membrane was sequentially probed for Cox-2 and β-actin. Representative results from three repeated experiments are shown. e 15d-PGJ2 synthesis in human colonic MΦs. Freshly isolated lamina propria MΦs (CD64± cells) were stimulated with LPS for 48 h. 15d-PGJ2 concentrations in supernatants were measured by EIA. Data of triplicate samples from 2 human subjects with SEM (* p < 0.05). f Real-time PCR results of TNF-α mRNA expression (mTNF-α) in mouse peritoneal MΦs with or without co-culturing with IECs in the Transwells. The relative expression of mouse TNF-α to β-actin after LPS stimulation is shown. Data from three independent experiments of triplicated samples with SEM (* p < 0.05).

Fig. 4

Epithelial TLR4 signaling inhibits degradation of PPARγ protein. a Western blot analysis of PPARγ in SW480-APC cells. SW480-APC cells were stimulated with LPS in conventional wells for the indicated periods. An equal amount of protein was loaded per lane. The membrane was sequentially probed for PPARγ and β-actin. Representative results from three repeated experiments are shown. b Real-time PCR results in PPARγ mRNA expression in SW480-APC cells grown in conventional wells. Cells were stimulated with LPS for the indicated period. The relative expression of human PPARγ to β-actin is shown. Data from three independent experiments of triplicate samples with SEM. n.s. = Nonsignificant. c Detection of ubiquitinated PPARγ by immunoprecipitation. Polarized SW480-APC cells were co-cultured with mouse peritoneal MΦs and basolaterally stimulated with LPS for the indicated periods. 400 μg of protein from SW480-APC cell lysates were immunoprecipitated with anti-PPARγ or control IgG and immunoblotted with HRP-conjugated anti-Ub monoclonal antibody (P4D1), followed by anti-PPARγ (bottom). Representative results from three repeated experiments are shown. Graph shows quantitative Ub band (each molecular weight level) intensity normalized with PPARγ band measured with ImageJ.

Fig. 5

MΦ TLR4 signaling induces activation of PPARγ in IECs during IEC-MΦ co-culture. a PPARγ activation in SW480-APC cells during co-culturing with MΦs. SW480-APC cells were transfected with a PPRE-luciferase reporter construct and co-cultured with MΦs in the Transwell system. LPS was added to the bottom wells for the indicated periods. PPARγ activation was expressed as percent increase of PPRE-luciferase activity over control (no LPS). Data from three independent experiments of duplicate samples with SEM (* p < 0.05). b PPARγ activation in SW480-APC cells without MΦ co-culture. SW480-APC cells were transfected with a PPRE-luciferase reporter construct and plated in the Transwell insert. Cells were basolaterally stimulated with LPS as indicated. PPARγ activation was expressed as percent increase of PPRE-luciferase activity over control (no LPS). Data from three independent experiments of duplicate samples with SEM. n.s. = Nonsignificant. c Involvement of MΦ TLR4 signaling in PPARγ activation of SW480-APC cells. SW480-APC cells were transfected with a PPRE-luciferase reporter construct and co-cultured with mouse peritoneal MΦs isolated from TLR4-/- mice or WT littermates in the Transwell system. LPS was added to the bottom wells. PPARγ activation was measured after 48 h as percent increase in PPRE-luciferase activity over control (no LPS). Data from three independent experiments of duplicate samples with SEM (* p < 0.05). d Immunofluorescent detection of cellular localization of PPARγ. Polarized SW480-APC cells were co-cultured with mouse peritoneal MΦs in the Transwell system and basolaterally stimulated with LPS for the indicated periods. PPARγ was labeled with Alexa Fluor 488 (green in the online version). Nuclei were stained with DAPI (blue in the online version). Original magnification ×40. Bars: 50 µm.

Fig. 6

Detection of functional PPRE motif in IL-10 promoter in human IEC. a hIL-10 promoter sequence in genomic DNA isolated from SW480-APC cells. PPRE motifs are underlined. b Detection of a functional PPRE element in hIL-10 promoter. SW480-APC cells were transfected with truncated hIL-10 promoter fragments of various lengths and co-cultured with mouse peritoneal MΦs in the Transwell system. LPS was added to the bottom wells and IL-10 promoter activity was measured after 72 h as percent increase of luciferase activity over control (empty vector). Data from three independent experiments of duplicate samples with SEM (** p < 0.01, *** p < 0.001). n.s. = Nonsignificant. c The binding of PPARγ to hIL-10 promoter. Polarized SW480-APC cells were co-cultured with mouse peritoneal MΦs in the Transwell system and basolaterally stimulated with LPS for 72 h as indicated. Nuclear protein was isolated from LPS-stimulated and non-stimulated SW480-APC cells and subjected to ChiP assay. Normal rabbit IgG was used for immunoprecipitation of control samples. Representative results from three repeated experiments are shown.

Fig. 7

The late induction of IL-10 in IEC-MΦ co-culture is epithelial PPARγ dependent. a IL-10 promoter activity. SW480-APC cells were transfected with −2,000-bp hIL-10 promoter and co-cultured with mouse peritoneal MΦs in the Transwell system. LPS was added to the bottom wells and IL-10 promoter activity was measured after 72 h as percent increase in luciferase activity over control (no LPS). The PPARγ antagonist SR202 (500 μM) was added to insert wells 90 min prior to LPS stimulation. Data from two independent experiments of duplicate samples with SEM (* p < 0.05). b Real-time PCR results of IL-10 mRNA expression in polarized SW480-APC cells in the presence or absence of PPARγ antagonist. Polarized SW480-APC cells with or without mouse peritoneal MΦs were basolaterally stimulated with LPS for 72 h. PPARγ antagonist SR202 (500 μM) was added to insert wells 90 min prior to LPS stimulation. The relative expression of hIL-10 to β-actin is shown. Data from three independent experiments of triplicate samples with SEM (* p < 0.05). n.s. = Nonsignificant. c The effect of SR202 on IL-10 production from polarized SW480-APC cell co-culturing with mouse peritoneal MΦs in the Transwell system. Cells were basolaterally stimulated with LPS for 72 h as indicated. PPARγ antagonist SR202 (500 μM) was added to insert wells 90 min prior to LPS stimulation. hIL-10 protein concentration in the bottom wells was measured by specific ELISA. Data from three independent experiments of duplicate samples with SEM (* p < 0.05). d The effect of SR202 on PPARγ activation in SW480-APC cells during co-culturing with MΦs. SW480-APC cells were transfected with a PPRE-luciferase reporter construct and co-cultured with mouse peritoneal MΦs in the Transwell system. LPS was added to the bottom wells for 48 h as indicated. The PPARγ antagonist SR202 (500 μM) was added to insert wells 90 min prior to LPS stimulation. PPARγ activation was expressed as percent increase in PPRE-luciferase activity over control (no LPS). Data from three independent experiments of duplicate samples with SEM (* p < 0.05).

Fig. 8

The role of epithelial PPARγ activation in IL-10 expression by IECs that are induced by MΦ TLR4 signaling. a Western blot analysis of PPARγ in Caco-2 cells. Caco-2 cells were stimulated with LPS in conventional wells for the indicated periods. An equal amount of protein was loaded per lane. The membrane was sequentially probed for PPARγ and β-actin. Representative results from three repeated experiments are shown. Graph shows ratios of PPARγ band intensity normalized with β-actin band measured with ImageJ. b Real-time PCR results of IL-10 mRNA expression in polarized Caco-2 cells. Polarized Caco-2 cells were co-cultured with mouse peritoneal MΦs in the Transwell system and basolaterally stimulated with LPS for the indicated periods. The relative expression of hIL-10 to β-actin is shown. Data from three independent experiments of triplicate samples with SEM (* p < 0.05). c IL-10 production from polarized Caco-2 cells with or without co-culturing with mouse peritoneal MΦs in the Transwell system. Cells were basolaterally stimulated with LPS for the indicated periods. hIL-10 protein concentration in the bottom wells was measured by specific ELISA. Data from three independent experiments of duplicate samples with SEM (* p < 0.05). d The role of PPARγ in the transcriptional activity of IL-10 in human IECs. Caco-2 cells were transfected with hIL-10 promoter construct together with siRNA against PPARγ and co-cultured with mouse peritoneal MΦs in the Transwell system. Control cells received IL-10 promoter construct and nonspecific siRNA. LPS was added to the bottom wells and IL-10 promoter activity was measured after 72 h as percent increase in luciferase activity over control (no LPS, control siRNA). The extent of PPARγ knockdown was assessed by Western blot. 15d-PGJ2 (5 ng/ml) was used as a positive control. Data from three independent experiments of duplicate samples with SEM (* p < 0.05). n.s. = Nonsignificant. e IL-10 production from polarized Caco-2 cells knocking down PPARγ. Caco-2 cells were transfected with siRNA against PPARγ and co-cultured with mouse peritoneal MΦs in the Transwell system. Control cells were transfected with nonspecific siRNA. Cells were basolaterally stimulated with LPS for 72 h as indicated. hIL-10 concentration in the bottom wells was measured by specific ELISA. Data from three independent experiments of duplicate samples with SEM (* p < 0.05). f The effect of MΦ Cox-2 induction on PPARγ activation in Caco-2 cells during co-culturing with MΦs. Caco-2 cells were transfected with a PPRE-luciferase reporter construct and co-cultured with mouse peritoneal MΦs in the Transwell system. LPS was added to the bottom wells for 48 h as indicated. Cox-2 antagonist NS398 (5 μM) was added to the bottom wells 90 min prior to LPS stimulation. PPARγ activation was expressed as percent increase in PPRE-luciferase activity over control (no LPS). Data from three independent experiments of duplicate samples with SEM (* p < 0.05).