IFNγ-dependent silencing of TFF1 during Helicobacter pylori infection

Abstract

Chronic Helicobacter pylori infection is the leading cause of intestinal-type adenocarcinoma, as prolonged Helicobacter colonization triggers chronic active gastritis, which may evolve into adenocarcinoma of the intestinal type. In this environment, cytokines play a significant role in determining the evolution of the infection. In combination with other factors (genetic, environmental and nutritional), the pro-inflammatory response may trigger pro-oncogenic mechanisms that lead to the silencing of tumour-suppressor genes, such as trefoil factor 1 (TFF1). The latter is known to play a protective role by maintaining the gastric mucosa integrity and retaining H. pylori in the mucus layer, preventing the progression of infection and, consequently, the development of gastric cancer (GC). Since TFF1 expression is reduced during chronic Helicobacter infection with a loss of gastric mucosa protection, we investigated the molecular pathways involved in this reduction. Specifically, we evaluated the effect of some pro-inflammatory cytokines on TFF1 regulation in GC and primary gastric cells by RT-qPCR and luciferase reporter assay analyses and the repressor role of the transcription factor C/EBPβ, overexpressed in gastric-intestinal cancer. Our results show that, among several cytokines, IFNγ stimulates C/EBPβ expression, which acts as a negative regulator of TFF1 by binding its promoter at three different sites.

Keywords: helicobacter; interferon-gamma; trefoil factor 1.

Figure 1.

Cytokines expression in PBMC or THP-1 cells infected by H. pylori. PBMC or THP-1 cells were infected with H. pylori at MOI 1 : 5 and cells were collected at 24-, 48- and 72 h post-infection. The indicated cytokines were measured by real-time PCR analysis. HPRT1 was used as a housekeeping gene. Graphs are representative of at least three independent experiments. Data are expressed as mean ± s.d. (t-test, *p-value ≤ 0.05, **p-value ≤ 0.01, ***p-value ≤ 0.001, ^#p-value ≤ 0.0001), and the mean of controls was set to 1.

Figure 2.

TFF1 expression is affected by conditioned media from H. pylori-infected immune cells (a) ELISA analysis of the indicated cytokines from PBMCs conditioned media. Data are expressed as mean ± s.d. (t-test, * p-value ≤ 0.05, ** p-value ≤ 0.01, *** p-value ≤ 0.001). (b,c) Western blot analysis of intracellular TFF1 protein in KATO III cells incubated with conditioned media (CM), respectively, from PBMCs (b) or THP-1 (c) cells infected with H. pylori at a MOI 1 : 5 for 72 h.

Figure 3.

TFF1 expression is downregulated by IFNγ. (a) Real-time PCR analysis of TFF1 in KATO III cells after 24 h incubation with TNFα (40 ng ml⁻¹), IFNγ (10 ng ml⁻¹) and IL1β (40 ng ml⁻¹) alone or in combination (mix). HPRT1 was used as a housekeeping gene. (b,c) Western blot analysis of intracellular TFF1 protein in KATO III cell line upon the above-described treatments and densitometric analysis of TFF1 signals (b) normalized to β-actin. (d,e) Western blot analysis of secreted TFF1 protein from the supernatants of KATO III treated as (a) and the densitometric analysis of TFF1. Images are representative of at least three independent experiments. Data are expressed as mean ± s.d. (t-test, **p-value ≤ 0.01, ***p-value ≤ 0.001), and the mean of the controls was set to 1.

Figure 4.

IFNγ reduces luciferase activity under TFF1 promoter control. (a) Luciferase reporter assays in KATO III cells transfected with pGL3-1kb-Luc plasmids, containing a fragment starting from −931 bp of TFF1 promoter upstream of a luciferase reporter gene and incubated with TNFα (40 ng ml⁻¹), IFNγ (10 ng ml⁻¹) and IL1β (40 ng ml⁻¹) alone or in combination (mix). (b) Luciferase reporter assays in KATO III cells transfected with different plasmids containing fragments of various lengths from −931 bp to −212 bp and stimulated with IFNγ for 24 h. Data are expressed as the mean of four independent experiments ± s.d. (t-test, **p-value ≤ 0.01, ***p-value ≤ 0.001), the mean of pGL3-0.2kb-Luc plasmid was set to 1.

Figure 5.

IFNγ induces C/EBPβ expression, which in turn represses TFF1. (a,b) KATO III cells were stimulated with 10 ng ml⁻¹ IFNγ, and at the indicated time points, TFF1 and C/EBPβ transcripts were measured by RTqPCR. HPRT1 was used as a housekeeping gene. Data are expressed as the mean of three independent experiments ± s.d. (t-test, **p-value ≤ 0.01, ***p-value ≤ 0.001). (c) C/EBPβ protein level was measured by western blot analysis in KATO III cells incubated with 10 ng ml⁻¹ IFNγ for 24 h. (d) Densitometric analysis of C/EBPβ from (c) normalized to β-actin. Images are representative of three independent experiments. Data are expressed as mean ± s.d. (t-test, * p-value ≤ 0.05). (e) KATO III cells were transfected with small interfering RNA (siRNA) control (Ctrl) or C/EBPβ and exposed to 1 ng ml⁻¹ IFNγ for 24 h. Protein expression of C/EBPβ and TFF1 was revealed by western blot analysis. β-actin served as a loading control.

Figure 6.

C/EBPβ binds to TFF1 upon IFNγ stimulation. (a) Schematic of C/EBPβ binding sites on TFF1 promoter. Site 1 (−216 bp) was identified by Sankpal et al. [24], while sites 2 (−483 bp) and 3 (−585 bp) are considered putative binding elements. (b–d) KATO III cells were stimulated with 10 ng ml⁻¹ IFNγ for 24 h, and the binding of C/EBPβ to TFF1 promoter was analysed by ChIP followed by RTqPCR analysis. Data are expressed as the mean of three independent experiments ± s.d. (t-test, *p-value ≤ 0.05).

Figure 7.

TFF1 expression in mucosoids is affected by cytokines during Helicobacter infection. (a) Immunofluorescence analysis of TFF1 (green) in ‘foveola’ (without Wnt3A/RSPO1, −W−R, a–c) and in ‘basal' cells (with Wnt3A/RSPO1, +W +R, d­–f) of mucosoids. Scale bars: 10 µm. (b) TFF1 and (c) IL8 expression level in mucosoids after 1, 3 and 5 days post H. pylori infection. HPRT1 was used as a housekeeping gene. Data are expressed as the mean of three independent experiments ± SD (t-test, **p-value ≤ 0.01, ****p-value ≤ 0.0001). (d) Microarray analysis of TFF1, TFF2 and TFF3 in mucosoids in +W +R conditions exposed to the indicated cytokines for 5 days. (e,f) Mucosoids were infected with H. pylori at MOI 50 and, the day after, exposed to the mixture of cytokines (TNFα, IL1β and IFNγ) for 5 days. (e) TFF1 and (f) IL8 transcripts were measured by RTqPCR. HPRT1 served as a housekeeping gene. Data are expressed as the mean of three independent experiments ± s.d. (t-test, **p-value ≤ 0.01, ***p-value ≤ 0.001).