Notch Signaling Pathway Is Activated by Sulfate Reducing Bacteria

Abstract

Sulfate Reducing Bacteria (SRB), usually rare residents of the gut, are often found in increased numbers (called a SRB bloom) in inflammatory conditions such as Inflammatory Bowel Disease (IBD), pouchitis, and periodontitis. However, the underlying mechanisms of this association remain largely unknown. Notch signaling, a conserved cell-cell communication pathway, is usually involved in tissue development and differentiation. Dysregulated Notch signaling is observed in inflammatory conditions such as IBD. Lipolysaccharide and pathogens also activate Notch pathway in macrophages. In this study, we tested whether Desulfovibrio, the most dominant SRB genus in the gut, may activate Notch signaling. RAW 264.7 macrophages were infected with Desulfovibrio vulgaris (DSV) and analyzed for the expression of Notch signaling pathway-related proteins. We found that DSV induced protein expression of Notch1 receptor, Notch intracellular domain (NICD) and p21, a downstream Notch target, in a dose-and time-dependent manner. DSV also induced the expression of pro-IL1β, a precursor of IL-1β, and SOCS3, a regulator of cytokine signaling. The gamma secretase inhibitor DAPT or Notch siRNA dampened DSV-induced Notch-related protein expression as well the expression of pro-IL1β and SOCS3. Induction of Notch-related proteins by DSV was not affected by TLR4 -IN -C34(C34), a TLR4 receptor antagonist. Additionally, cell-free supernatant of DSV-infected macrophages induced NICD expression in uninfected macrophages. DSV also activated Notch pathway in the human epithelial cell line HCT116 and in mouse small intestine. Thus, our study uncovers a novel mechanism by which SRB interact with host cells by activating Notch signaling pathway. Our study lays a framework for examining whether the Notch pathway induced by SRB contributes to inflammation in conditions associated with SRB bloom and whether it can be targeted as a therapeutic approach to treat these conditions.

Keywords: DAPT; Desulfovibrio vulgaris (DSV); NICD; Notch1; SOCS3; pro-IL-1β; sulfate reducing bacteria (SRB).

Figure 1

DSV induces dose and time-dependent activation of Notch1 signaling markers, SOCS3, and pro-IL-1β expression (A) RAW cells (8x10⁵) were infected with DSV at MOI 5, 20 or 50 for 7 hours. Cells were lysed and protein lysate was prepared. Fifty μg of protein lysate was separated on SDS-PAGE and analyzed for Notch1, NICD, SOCS3, and IL-1β by Western blotting. Actin was used as a loading control. (B–E) Quantification of Western blots. Blots were quantified with ImageJ by analyzing the ratio of protein of interest/Actin. Data represents Mean ± SEM from at least three independent experiments. Values were normalized to control. (F) Cells were infected with DSV (MOI 20) for 0.5, 2, 4, and 7 hours. Fifty μg of protein lysate was separated on SDS-PAGE and analyzed for Notch1, NICD, SOCS3, and IL-1β by Western blotting. Actin was used as a loading control. (G–J) Quantification of Western blots. Blots were quantified with ImageJ by analyzing the ratio of protein of interest/Actin. Values were normalized to control. Data represents Mean ± SEM from at least three independent experiments. One-way ANOVA was used to determine the statistical significance. Values were compared to control, with a post-hoc Dunnett’s test. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2

DSV differentially induces gene expression of Notch ligands and receptors RAW cells were treated with DSV (MOI 20) for 7hrs and harvested, RNA was isolated and cDNA synthesized. QPCR was carried out to determine the gene expression of (A) DLL1, (B) DLL3, (C) DLL4, (D) Jag1, (E) Jag2, (F) Notch1, (G), Notch2, (H) Notch3, and (I) Notch4 using Taqman probes for respective genes. Relative fold change expression in DSV treated cells compared to untreated control cells was calculated using 2^-ddct method using 18s gene expression as the housekeeping control. A two-tailed t-test was used to determine the statistical difference between control and DSV-treated cells. *p < 0.05, ***p < 0.001.

Figure 3

DSV-induced Notch signaling, SOCS3, and pro-IL-1β expression is inhibited by gamma secretase inhibitor DAPT (A) RAW cells (8x10⁵) were treated with various concentrations of DAPT (10, 25, and 50 µm in DMSO) overnight. The following day, cells were infected with DSV (MOI 20) for 7 hours. Cells were lysed and protein lysate was prepared. Fifty μg of protein lysate was separated on SDS-PAGE and analyzed for Notch1, NICD, SOCS3, IL-1β, and p21 by Western blotting. Actin was used as a loading control. (B–F) Quantification of Western blots. Blots were quantified with ImageJ by analyzing the ratio of protein of interest/Actin. Data represents Mean ± SEM from at least three independent experiments. One-way ANOVA was used to determine the statistical significance. Values were compared to control, with a post-hoc Dunnett’s test. **p < 0.01 and ***p < 0.001.

Figure 4

DSV-induced Notch signaling, SOCS3, and IL-1β expression is inhibited by Notch1siRNA (A) RAW cells were transfected with either scrambled (Scr) or siRNA against Notch1 (siNotch1) by electroporation. After incubation for 24 hours, cells were removed and re-plated at a density of 8x10⁵ and further incubated for 24 hours. Following day (total of 48 hrs transfection), cells were infected with DSV (MOI 20) for 7 hours. Cells were lysed and protein lysate was prepared. Fifty μg of protein lysate was separated on SDS-PAGE and analyzed for Notch1, NICD, SOCS3, IL-1β, and p21 by Western blotting. Actin was used as a loading control. (B–F) Quantification of Western blots. Blots were quantified with ImageJ by analyzing the ratio of protein of interest/Actin. Data represents Mean ± SEM from at least three independent experiments. One-way ANOVA was used to determine the statistical significance. Values were compared to Scr+ DSV, with a post-hoc Dunnett’s test. *p < 0.05, **p < 0.01.

Figure 5

DSV-Induced Notch Activation occurs independent of TLR4 activation (A) RAW cells (8x10⁵) were first treated with 15µm C34, A TLR4 inhibitor for 30 mins prior to infection with DSV. Cells were harvested and protein lysate was prepared. Fifty μg of protein lysate was separated on SDS-PAGE and analyzed for NICD, SOCS3, and p21 expression by Western blotting. Actin was used as a loading control. (B–D) Quantification of Western blots. Blots were quantified with ImageJ by analyzing the ratio of protein of interest/Actin. Data represents Mean ± SEM from at least three independent experiments. Values were normalized to control. Ϯp > 0.05.

Figure 6

Schematic representation of the methodology for testing paracrine effect of soluble factors in DSV-infected cells on uninfected recipient cells.

Figure 7

Paracrine activation of Notch in recipient cells by culture supernatant of DSV-treated RAW cells (A) Flow chart of the experimental strategy to examine the paracrine effects of DSV-Notch signaling on uninfected target cells. See Figures 6 . (B) 8x10⁵ cells were plated the day before infection. Cells were infected with DSV (MOI 20) for 7 hours. Culture supernatants were collected and the infected cells were harvested and lysed. Control Sup, DSV-RAW cell sup, or DSV+RAW cell sup was passed through a 0.2 µm filter and added to a fresh plate of uninfected recipient cells overnight. The following day, the recipient cells were harvested and lysed. Fifty μg of protein lysate was separated on SDS-PAGE and analyzed for NICD expression by Western blotting. Actin was used as a loading control. (C) Quantification of Western blots. Blots were quantified with ImageJ by analyzing the ratio of protein of interest/Actin. Data represents Mean ± SEM from at least three independent experiments. One-way ANOVA was used to determine the statistical significance. Values were compared to control sup, with a post-hoc Dunnett’s test. *p < 0.05. (D) Same as in (A) but in this experiment, recipient cells were pre-treated with various concentrations of DAPT overnight. Medium was removed and replaced with either control sup, DSV-RAW cell sup or with DSV+RAW cell sup for overnight incubation. Fresh DAPT was again added to the cells along with the culture supernatants. (E) Quantification of Western blots. Blots were quantified with ImageJ by analyzing the ratio of protein of interest/Actin. Data represents Mean ± SEM from at least three independent experiments. One-way ANOVA was used to determine the statistical significance. Values were compared to control sup, with a post-hoc Dunnett’s test. ***p < 0.001.

Figure 8

DSV induces Notch activation in epithelial cells (A) HCT116 cells (8x10⁵) were infected with DSV at MOI 20 for 7 hours. Cells were lysed and protein lysate was prepared. Fifty μg of protein lysate was separated on SDS-PAGE and analyzed for NICD and Actin by Western blotting. (B) Quantification of Western blots. Blots were quantified with ImageJ by analyzing the ratio of NICD/Actin. Data represents Mean ± SEM from at least three independent experiments. Values were normalized to control. **p < 0.01.

Figure 9

DSV induces NICD activation in the mouse small intestine Mice were orally gavaged with either PBS or DSV (10⁹). One hour later, mice were euthanized and 1/3^rd parts of small intestinal tissue corresponding to Prox (duodenum), Mid (Jejunum), and Distal (ileum) regions were collected in Trizol. Protein samples (10µg) were prepared and analyzed by Western blotting for NICD expression. Actin was used as a loading control. (A) NICD expression in protein samples from 4 control and 5 DSV-treated tissues (B–D) Quantification of Western blots. Blots were quantified with ImageJ by analyzing the ratio of NICD/Actin. Data represents Mean ± SEM. **p < 0.01, Ϯp > 0.05.