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, 10 (10), 748

IL-6-driven FasL Promotes NF-κBp65/PUMA-mediated Apoptosis in Portal Hypertensive Gastropathy

Affiliations

IL-6-driven FasL Promotes NF-κBp65/PUMA-mediated Apoptosis in Portal Hypertensive Gastropathy

Siwei Tan et al. Cell Death Dis.

Abstract

Mucosal epithelial apoptosis with non-specific inflammation is an essential pathological characteristic in portal hypertensive gastropathy (PHG). However, whether a coordinated crosstalk between myeloid cells and epithelial cells involved in PHG remains unclear. IL-6, which is induced in the mucosa of PHG patients and mice, promotes FasL production via enhancing NF-κBp65 activation in myeloid cells, while blockage of IL-6 signaling by Tocilizumab or deletion of NF-κBp65 in myeloid cells attenuates the inflammatory response and Fas/FasL-mediated epithelial apoptosis in PHG. IL-6-driven FasL from myeloid cells combines with epithelial Fas receptor to encourage NF-κBp65/PUMA-mediated epithelial apoptosis in PHG, and inhibition of NF-κBp65 or knockout of PUMA alleviates Fas/FasL-mediated epithelial apoptosis in PHG. These results indicate that IL-6 drives FasL generation via NF-κBp65 in myeloid cells to promote Fas/NF-κBp65/PUMA-mediated epithelial apoptosis in PHG, and this coordinated crosstalk between myeloid cells and epithelial cells may provide a potential therapeutic target for PHG.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. IL-6 involved in PHG.
a, b Expressions of indicated inflammatory cytokines in the related gastric mucosa were analyzed. β-actin was used as an internal control. n = 6 in each group, values are presented as mean ± SEM. Bonferroni’s comparison post hoc test. *P < 0.05 versus uninvolved tissues or SO (sham operation) mice, respectively. c Two-dimensional hierarchical clustering results for the gene of IL-6 superfamily members between PHG patients and healthy volunteers (n = 6 per group). d The fold changes and P value of the indicated mRNA levels in PHG tissues relative to normal (uninvolved) tissues from microarray experiment were represented. e Endoscopic imaging and IL-6 immunohistochemistry (IHC) staining of uninvolved normal gastric mucosal tissue and gastropathic mucosal tissue from PHG patient were presented (brown, × 400, n = 6 per group). f IL-6 staining and gastric imaging in the indicated sections from SO and PVL mice (brown, × 400, n = 6 per group). g, h IL-6 and IL-6R levels in the indicated gastric mucosa were determined by western blotting in three pairs of different representative specimens. β-actin was used as the loading control. n = 6 per group
Fig. 2
Fig. 2. Inhibition of IL-6 signaling attenuated inflammatory response and Fas-mediated epithelial apoptosis in PHG.
a The indicated inflammatory cell staining and mucosal TUNEL staining in the tissues were analyzed (brown, × 400, n = 6 per group). b The gastric injury index (left panel), CD68 index (macrophage), CD19 index (B cell), CD3 index (T cell), MPO index (neutrophil), and the apoptosis index (TUNEL staining) analyzed from (a). n = 6 in each group, values are presented as mean ± SEM. c Gastric mucosal Fas, FasL and cleaved caspase-3 expression were evaluated by western blotting. β-actin was used as the loading control. n = 6 per group. d Western blotting analyses revealed that the levels of TNF-α/TNFR1 and TRAIL/DR4/DR5 in PVL mice were not affected by Tocilizumab administration. β-actin was used as the loading control. n = 6 per group
Fig. 3
Fig. 3. IL-6 drove epithelial apoptosis via upregulating myeloid FasL.
a Immunohistochemical staining showed that IL-6 administration upregulated the expression of Fas and FasL in mice gastric mucosa (n = 6 per group). b Fas and Fasl mRNA from isolated primary epithelial and myeloid cells analyzed by PCR. β-actin was used as an internal control. n = 6 in each group, values are presented as mean ± SEM. Bonferroni’s comparison post hoc test. c The viability of primary myeloid cells and epithelial cells isolated from p65f/f (floxed p65) mice or p65ΔM/ΔM (myeloid cells specific NF-κBp65 deletion) mice were assayed by the Cell Counting Kit-8 (CCK-8) analysis, respectively. n = 6 per group. d FasL, but not Fas and cleaved caspase-3, was induced in primary myeloid cells of IL-6-treated mice (left panel). Fas and cleaved caspase-3, but not FasL, were induced in primary epithelial cells of IL-6-treated mice (right panel). n = 6 in each group, values are presented as mean ± SEM. e Expressions of Fas, FasL and cleaved caspase-3 in GES-1 cells analyzed by western blotting. β-actin was used as the loading control. n = 6 in each group, values are presented as mean ± SEM. f Immunofluorescence staining of Fas (green) and TUNEL staining indicated IL-6 could not promote primary epithelial cells apoptosis. Cell nuclei (blue) were counterstained by DAPI (× 800). The Fas index and the apoptotic index were also represented. n = 6 in each group, values are presented as mean ± SEM. NS, no significance
Fig. 4
Fig. 4. IL-6 upregulated FasL levels via NF-κBp65 in myeloid cells in PHG.
a FasL and NF-κBp65 phosphorylation (NF-κBp-p65, as p-p65), rather than Fas, were induced in primary myeloid cells isolated from PVL-treated mice. β-actin was used as the loading control. n = 6 per group. b The activity of STAT3 and ERK1/2 were detected in primary myeloid cells isolated from SO (sham operation)- and PVL-treated mice. β-actin was used as the loading control. n = 6 per group. c NF-κBp65 deficiency in myeloid cells downregulated the expression of FasL, without affecting the level of IL-6 in the gastric mucosa of PVL mice (brown, × 400, n = 6 per group). d The levels of NF-κBp-p65, NF-κBp65, IκBα, FasL and Fas in the primary myeloid cells dissociated from PVL-treated mice were determined. p65f/f, floxed p65 mice. p65ΔM/ΔM, myeloid cells specific NF-κBp65 deletion mice. β-actin was used as the loading control. n = 6 per group
Fig. 5
Fig. 5. IL-6-driven FasL contributed to epithelial apoptosis via Fas in PHG.
a Fas, FasL staining and TUNEL staining from the indicated gastric tissues were revealed (brown, × 400, n = 6 per group). b Double staining of Fas (green) and TUNEL (red) indicated that Fas-mediated apoptosis contributed to PHG, nuclei (blue) were counterstained with DAPI (× 800, n = 6 per group). c Co-staining of Fas (green) and cytokeratin (red) demonstrated that Fas mainly located in the epithelial cells of both PHG patients and mice, nuclei (blue) were counterstained with DAPI (× 800, n = 6 per group). d Flow cytometric analysis of primary myeloid and epithelial cells isolated from SO (sham operation)- and PVL-treated mice with anti-Fas or anti-FasL antibodies (n = 6 per group). e Western blotting showed that Fas, FasL and cleaved caspase-3 were enhanced in total mucosa of PHG sections (left panel). The levels of Fas, FasL and cleaved caspase-3 in the primary myeloid and epithelial cells dissociated from PHG patients and healthy volunteers (as Un, uninvolved) were determined (right panel). β-actin was used as the loading control. n = 6 per group. f Fas, FasL and cleaved caspase-3 were enhanced in PVL-treated sections. The expressions of Fas, FasL and cleaved caspase-3 in the primary myeloid and epithelial cells dissociated from SO- and PVL-treated mice were determined (right panel). β-actin was used as the loading control. n = 6 per group
Fig. 6
Fig. 6. NF-κBp65 deficiency in gastric myeloid cells attenuated Fas-mediated epithelial apoptosis in PHG.
a Gastric mucosal apoptosis was depressed in PVL-treated p65ΔM/ΔM (myeloid cells specific NF-κBp65 deletion) mice compared with p65f/f (floxed p65) mice (TUNEL staining, × 400, n = 6 per group). b Double staining of cytokeratin (green) and TUNEL (apoptotic cells, red) indicated that gastric apoptotic cells mainly located in the epithelial cells of the gastric mucosa from PVL mice (upper, × 400; lower panel, × 800, n= 6 per group). c The apoptotic index from TUNEL staining was presented. n = 6 in each group, values are presented as mean ± SEM. *P < 0.05 versus SO (sham operation) mice, #P < 0.05 versus PVL-treated p65f/f mice. d Western blotting analysis demonstrated that deletion of NF-κBp65 in myeloid cells blocked Fas, cleaved caspase-3 levels and NF-κBp65 phosphorylation in the gastric epithelial cells from PVL-treated mice. β-actin was used as the loading control. n = 6 per group
Fig. 7
Fig. 7. Fas/FasL promoted epithelial apoptosis via NF-κBp65 in PHG.
a Representative images of NF-κBp-p65 and TUNEL staining from the indicated tissues (brown, × 400, n = 6 per group). b Western blotting represented that NF-κBp-p65 was upregulated in gastropathic mucosal tissue from PHG patients. β-actin was used as the loading control. n = 6 per group. c The NF-κB inhibitor BAY inhibited mucosal apoptosis both in PVL-treated p65f/f and p65ΔM/ΔM mice, without affecting that in SO (sham operation) mice (TUNEL staining, brown, × 400, n = 6 per group). d BAY inhibited caspase-3 cleavage, Fas, FasL, mucosal apoptosis and NF-κBp65 phosphorylation in PVL-treated p65f/f mice. β-actin was used as the loading control. n = 6 per group. e Double staining of NF-κBp-p65 (green) and TUNEL (red), nuclei (blue) were counterstained with DAPI (× 800, n = 6 per group). f The apoptosis index was determined from (c) (upper panel). n = 6 in each group, values are presented as mean ± SEM. *P < 0.05 versus p65f/f mice without BAY administration, #P < 0.05 versus p65ΔM/ΔM mice without BAY administration
Fig. 8
Fig. 8. PUMA participated in Fas/FasL/NF-κBp65-mediated epithelial apoptosis in PHG.
a Real-time PCR suggested PUMA mRNA expression was increased by fivefold in PHG and fourfold in PVL-treated mice, compared with their control group, respectively. β-actin was used as an internal control. n = 6 in each group, values are presented as mean ± SEM. Bonferroni’s comparison post hoc test. b Immunohistochemical staining of PUMA was presented (brown, upper panel, × 400, n = 6 per group). The levels of PUMA in the indicated tissues were analyzed by western blotting (lower panel). β-actin was used as the loading control. c PUMA staining demonstrated that deletion of NF-κBp65 in myeloid cells of PUMA-WT mice repressed the expression of PUMA (brown, × 400, n= 6 per group). d Western blotting indicated that deletion of NF-κBp65 in myeloid cells of PUMA-WT mice attenuated PUMA-mediated apoptosis (left panel). β-actin was used as the loading control. n = 6 per group. e Immunohistochemical staining showed deletion of PUMA in p65f/f mice attenuated PVL-induced apoptosis (TUNEL staining) without affecting the station of NF-κBp65 activity (p-p65 staining) and Fas level (brown, × 400, n = 6 per group). f, g Western blotting depicted that PUMA deficiency in p65f/f mice suppressed cleaved caspase-3 expression without influencing the levels of Fas, FasL and NF-κBp65 activity in PVL-treated mice. β-actin was used as the loading control. n = 6 per group. h The gastric injury index analysis represented that gastric mucosal injury was attenuated in PVL-treated p65f/f/PUMA-KO mice compared with p65f/f/PUMA-WT mice. n = 6 in each group, values are presented as mean ± SEM. *P < 0.05 versus SO mice, #P < 0.05 versus PVL-treated p65f/f/PUMA-WT mice
Fig. 9
Fig. 9. IL-6-driven FasL in myeloid cells facilitated PUMA-mediated epithelial apoptosis in vitro.
a Schematic diagram of the co-culture experiments with primary myeloid cells and epithelial cells isolated from mice. b The concentration of FasL in the indicated medium of (a) was detected with ELISA (left panel, n = 6 per group). Values are presented as mean ± SEM. *P < 0.05 versus the medium of PBS-treated cells, #P < 0.05 versus the medium of IL-6-treated p65f/f/PUMA-WT cells. Expressions of the related proteins of primary myeloid cells were detected by western blotting (right panel, n = 6 per group). c The extent of primary epithelial cells apoptosis from the co-culture experiments was preformed using an Annexin V-PE/7-AAD staining kit (Flow cytometric analysis). d The indicated proteins from primary epithelial cells in co-culture experiments were presented, and deletion of NF-κBp65 in myeloid cells of PUMA-WT mice repressed Fas, NF-κBp-p65, PUMA and cleaved caspase-3, without affecting Bid and caspase-8 cleavage, in epithelial cells under IL-6 treatment in co-culture (upper panel). The epithelial cells viability was analyzed by CCK-8 assay (lower panel). *P < 0.05 versus the medium of PBS-treated cells. e Western blotting depicted IL-6 treatment did not influenced the levels of Fas, FasL, PUMA and cleaved caspase-3 of the isolated primary epithelial cells. β-actin was used as the loading control. n = 6 per group. f FasL treatment enhanced the expressions of Fas, NF-κBp-p65, PUMA and cleaved caspase-3 of the isolated primary epithelial cells. β-actin was used as the loading control. n = 6 per group. g Mitochondrial and cytosolic fractions were analyzed for cytochrome c by western blotting (n = 6 per group). β-actin and COX IV were used as the loading control of cytosolic and mitochondrial fractions, respectively

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