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Fas/FasL pathway participates in regulation of antiviral and inflammatory response during mousepox infection of lungs.

Bień K, Sokołowska J, Bąska P, Nowak Z, Stankiewicz W, Krzyzowska M - Mediators Inflamm. (2015)

Bottom Line: In this study, we examined the role of Fas/FasL pathway in inflammatory and antiviral response in lungs using a mousepox model applied to C57BL6/J, B6.Experiments in vitro demonstrated that ECTV-infected cultures of epithelial cells, but not macrophages, upregulate Fas and FasL and are susceptible to Fas-induced apoptosis.Our study demonstrates that Fas/FasL pathway during ECTV infection of the lungs plays an important role in controlling local inflammatory response and mounting of antiviral response.

View Article: PubMed Central - PubMed

Affiliation: Military Institute of Hygiene and Epidemiology, Kozielska 4, 01-163 Warsaw, Poland.

ABSTRACT
Fas receptor-Fas ligand (FasL) signalling is involved in apoptosis of immune cells as well as of the virus infected target cells but increasing evidence accumulates on Fas as a mediator of apoptosis-independent processes such as induction of activating and proinflammatory signals. In this study, we examined the role of Fas/FasL pathway in inflammatory and antiviral response in lungs using a mousepox model applied to C57BL6/J, B6. MRL-Faslpr/J, and B6Smn.C3-Faslgld/J mice. Ectromelia virus (ECTV) infection of Fas- and FasL-deficient mice led to increased virus titers in lungs and decreased migration of IFN-γ expressing NK cells, CD4+ T cells, CD8+ T cells, and decreased IL-15 expression. The lungs of ECTV-infected Fas- and FasL-deficient mice showed significant inflammation during later phases of infection accompanied by decreased expression of anti-inflammatory IL-10 and TGF-β1 cytokines and disturbances in CXCL1 and CXCL9 expression. Experiments in vitro demonstrated that ECTV-infected cultures of epithelial cells, but not macrophages, upregulate Fas and FasL and are susceptible to Fas-induced apoptosis. Our study demonstrates that Fas/FasL pathway during ECTV infection of the lungs plays an important role in controlling local inflammatory response and mounting of antiviral response.

No MeSH data available.


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ECTV-infected macrophages and epithelial cells show different response to Fas-induced apoptosis. (a) Percentages of ECTV-infected cells in Hepa 1–6 epithelial cell cultures and bone marrow derived macrophages (BMMF) at 24 h p.i. (b) Percentage of Fas- and FasL-positive cells in epithelial Hepa 1–6 and BMMF cultures at 24 h of ECTV infection. Percentage of apoptotic (annexin V-positive) cells in epithelial Hepa 1–6 (c) and BMMF cultures (d), infected or not with ECTV and exposed or not to anti-Fas cytotoxic antibody (10 mg/mL). (e) Percentage of apoptotic cells in the cocultures of epithelial Hepa 1–6 and macrophage cells infected or not with ECTV and exposed to anti-FasL-blocking antibody (10 μg/mL). The bars represent the mean from 3 separate experiments (N = 3) ± SEM.  *Significant differences with P ≤ 0.05 and **P ≤ 0.01.
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fig5: ECTV-infected macrophages and epithelial cells show different response to Fas-induced apoptosis. (a) Percentages of ECTV-infected cells in Hepa 1–6 epithelial cell cultures and bone marrow derived macrophages (BMMF) at 24 h p.i. (b) Percentage of Fas- and FasL-positive cells in epithelial Hepa 1–6 and BMMF cultures at 24 h of ECTV infection. Percentage of apoptotic (annexin V-positive) cells in epithelial Hepa 1–6 (c) and BMMF cultures (d), infected or not with ECTV and exposed or not to anti-Fas cytotoxic antibody (10 mg/mL). (e) Percentage of apoptotic cells in the cocultures of epithelial Hepa 1–6 and macrophage cells infected or not with ECTV and exposed to anti-FasL-blocking antibody (10 μg/mL). The bars represent the mean from 3 separate experiments (N = 3) ± SEM.  *Significant differences with P ≤ 0.05 and **P ≤ 0.01.

Mentions: To study the role of Fas-dependent apoptotic response during ECTV infection, we employed an in vitro model consisting of mouse Hepa 1–4 epithelial cell culture and bone marrow derived macrophage cultures (BMMF) infected with ECTV for 24 h. Both cell cultures infected with MOI = 2 of ECTV showed approximately 60% of infected cells (Figure 5(a)). Upon infection with ECTV, epithelial cell cultures significantly upregulated Fas and FasL expression (P ≤ 0.05) (Figure 5(b)), while mouse macrophages infected with ECTV significantly decreased Fas expression (P ≤ 0.001) (Figure 5(b)). To assess the sensitivity of ECTV-infected epithelial cells and macrophages to Fas-induced apoptosis we used an anti-mouse Fas cytotoxic antibody (Jo-1 clone). In both the Hepa 1–6 epithelial cell cultures and macrophages, addition of the anti-Fas antibody to uninfected cultures resulted in an increased percentage of annexin V-positive cells after 24 h (P ≤ 0.05) (Figures 5(c) and 5(d)). Addition of anti-Fas antibody to ECTV-infected cultures significantly increased Fas-mediated apoptosis but only in epithelial cells (P ≤ 0.05) (Figures 5(c) and 5(d)). In order to test the role of Fas-induced apoptosis in the lungs during ECTV infection we constructed a model of epithelial cells cocultured with macrophages in the presence of FasL-blocking antibody (clone MFL4) (Figure 5(e)). At 24 h of infection in the presence of FasL-blocking antibody, only epithelial cells showed a significant decrease in the percentage of apoptotic cells (P ≤ 0.001) (Figure 5(e)). ECTV infection of cocultures led to a significant decrease in production of TNF-α, CXCL1, and CXCL9 (P ≤ 0.05) (Table 1). However, blocking of Fas-FasL interaction between macrophages and epithelial cells further downregulated CXCL1 and CXCL9 production (P ≤ 0.05) (Table 1).


Fas/FasL pathway participates in regulation of antiviral and inflammatory response during mousepox infection of lungs.

Bień K, Sokołowska J, Bąska P, Nowak Z, Stankiewicz W, Krzyzowska M - Mediators Inflamm. (2015)

ECTV-infected macrophages and epithelial cells show different response to Fas-induced apoptosis. (a) Percentages of ECTV-infected cells in Hepa 1–6 epithelial cell cultures and bone marrow derived macrophages (BMMF) at 24 h p.i. (b) Percentage of Fas- and FasL-positive cells in epithelial Hepa 1–6 and BMMF cultures at 24 h of ECTV infection. Percentage of apoptotic (annexin V-positive) cells in epithelial Hepa 1–6 (c) and BMMF cultures (d), infected or not with ECTV and exposed or not to anti-Fas cytotoxic antibody (10 mg/mL). (e) Percentage of apoptotic cells in the cocultures of epithelial Hepa 1–6 and macrophage cells infected or not with ECTV and exposed to anti-FasL-blocking antibody (10 μg/mL). The bars represent the mean from 3 separate experiments (N = 3) ± SEM.  *Significant differences with P ≤ 0.05 and **P ≤ 0.01.
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Related In: Results  -  Collection

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fig5: ECTV-infected macrophages and epithelial cells show different response to Fas-induced apoptosis. (a) Percentages of ECTV-infected cells in Hepa 1–6 epithelial cell cultures and bone marrow derived macrophages (BMMF) at 24 h p.i. (b) Percentage of Fas- and FasL-positive cells in epithelial Hepa 1–6 and BMMF cultures at 24 h of ECTV infection. Percentage of apoptotic (annexin V-positive) cells in epithelial Hepa 1–6 (c) and BMMF cultures (d), infected or not with ECTV and exposed or not to anti-Fas cytotoxic antibody (10 mg/mL). (e) Percentage of apoptotic cells in the cocultures of epithelial Hepa 1–6 and macrophage cells infected or not with ECTV and exposed to anti-FasL-blocking antibody (10 μg/mL). The bars represent the mean from 3 separate experiments (N = 3) ± SEM.  *Significant differences with P ≤ 0.05 and **P ≤ 0.01.
Mentions: To study the role of Fas-dependent apoptotic response during ECTV infection, we employed an in vitro model consisting of mouse Hepa 1–4 epithelial cell culture and bone marrow derived macrophage cultures (BMMF) infected with ECTV for 24 h. Both cell cultures infected with MOI = 2 of ECTV showed approximately 60% of infected cells (Figure 5(a)). Upon infection with ECTV, epithelial cell cultures significantly upregulated Fas and FasL expression (P ≤ 0.05) (Figure 5(b)), while mouse macrophages infected with ECTV significantly decreased Fas expression (P ≤ 0.001) (Figure 5(b)). To assess the sensitivity of ECTV-infected epithelial cells and macrophages to Fas-induced apoptosis we used an anti-mouse Fas cytotoxic antibody (Jo-1 clone). In both the Hepa 1–6 epithelial cell cultures and macrophages, addition of the anti-Fas antibody to uninfected cultures resulted in an increased percentage of annexin V-positive cells after 24 h (P ≤ 0.05) (Figures 5(c) and 5(d)). Addition of anti-Fas antibody to ECTV-infected cultures significantly increased Fas-mediated apoptosis but only in epithelial cells (P ≤ 0.05) (Figures 5(c) and 5(d)). In order to test the role of Fas-induced apoptosis in the lungs during ECTV infection we constructed a model of epithelial cells cocultured with macrophages in the presence of FasL-blocking antibody (clone MFL4) (Figure 5(e)). At 24 h of infection in the presence of FasL-blocking antibody, only epithelial cells showed a significant decrease in the percentage of apoptotic cells (P ≤ 0.001) (Figure 5(e)). ECTV infection of cocultures led to a significant decrease in production of TNF-α, CXCL1, and CXCL9 (P ≤ 0.05) (Table 1). However, blocking of Fas-FasL interaction between macrophages and epithelial cells further downregulated CXCL1 and CXCL9 production (P ≤ 0.05) (Table 1).

Bottom Line: In this study, we examined the role of Fas/FasL pathway in inflammatory and antiviral response in lungs using a mousepox model applied to C57BL6/J, B6.Experiments in vitro demonstrated that ECTV-infected cultures of epithelial cells, but not macrophages, upregulate Fas and FasL and are susceptible to Fas-induced apoptosis.Our study demonstrates that Fas/FasL pathway during ECTV infection of the lungs plays an important role in controlling local inflammatory response and mounting of antiviral response.

View Article: PubMed Central - PubMed

Affiliation: Military Institute of Hygiene and Epidemiology, Kozielska 4, 01-163 Warsaw, Poland.

ABSTRACT
Fas receptor-Fas ligand (FasL) signalling is involved in apoptosis of immune cells as well as of the virus infected target cells but increasing evidence accumulates on Fas as a mediator of apoptosis-independent processes such as induction of activating and proinflammatory signals. In this study, we examined the role of Fas/FasL pathway in inflammatory and antiviral response in lungs using a mousepox model applied to C57BL6/J, B6. MRL-Faslpr/J, and B6Smn.C3-Faslgld/J mice. Ectromelia virus (ECTV) infection of Fas- and FasL-deficient mice led to increased virus titers in lungs and decreased migration of IFN-γ expressing NK cells, CD4+ T cells, CD8+ T cells, and decreased IL-15 expression. The lungs of ECTV-infected Fas- and FasL-deficient mice showed significant inflammation during later phases of infection accompanied by decreased expression of anti-inflammatory IL-10 and TGF-β1 cytokines and disturbances in CXCL1 and CXCL9 expression. Experiments in vitro demonstrated that ECTV-infected cultures of epithelial cells, but not macrophages, upregulate Fas and FasL and are susceptible to Fas-induced apoptosis. Our study demonstrates that Fas/FasL pathway during ECTV infection of the lungs plays an important role in controlling local inflammatory response and mounting of antiviral response.

No MeSH data available.


Related in: MedlinePlus