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. 2015:2015:281613.
doi: 10.1155/2015/281613. Epub 2015 Mar 22.

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

Karolina Bień  1 , Justyna Sokołowska  2 , Piotr Bąska  3 , Zuzanna Nowak  4 , Wanda Stankiewicz  1 , Malgorzata Krzyzowska  1
Affiliations

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

Karolina Bień et al. Mediators Inflamm. 2015.

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.

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Figures

Figure 1
Figure 1
Lack of Fas and FasL expression results in an increased infection burden during mousepox infection. (a) Fas and FasL expression in the lungs of C57BL/6 mice uninfected and ECTV-infected at the 7th d.p.i. Brown color indicates a positive reaction (Fas+ or FasL−), while blue color corresponds with hematoxylin positive nuclei. (b, c) C57BL/6 (WT), B6. MRL-Faslpr/J (Fas−), and B6Smn.C3-Faslgld/J (FasL−) mice (n = 70 in each group) were infected with 1 × 103 PFU of ECTV-MOS and monitored for 14 days. Kaplan-Meier survival analysis using the log-rank test (b) and (c) ECTV titers in lungs. Asterisks along the lines indicate statistical differences between the mice strains. The bars represent the mean from 5 separate experiments ± SEM.  **Significant differences with P ≤ 0.001 in comparison to Fas- and FasL-deficient mice.
Figure 2
Figure 2
Lack of Fas or FasL leads to disturbances in the inflammatory response. (a) Histological analysis of lungs isolated from ECTV-infected Fas (−), FasL (−), and WT (C57BL/6) mice at 7 and 10 days of infection and uninfected controls. The nuclei were counterstained with Harris haematoxylin (violet) (×40 and 100). Total counts of alveolar macrophages (b) and inflammatory monocytes (b) in cell suspensions prepared from the lungs isolated from Fas (−), FasL (−), and WT (C57BL/6) mice at 3, 7, 10, and 14 days of ECTV infection and from control, uninfected mice. The bars represent the mean from 5 separate experiments ± SEM.  *Significant differences with P ≤ 0.05 and ** P ≤ 0.01 in comparison to wild-type mice.
Figure 3
Figure 3
Lack of Fas or FasL leads to disturbances in antiviral immune response. Total counts of NK cells (a) NK/IFN-γ+ cells (b), CD4+ T cells (c), CD4+/IFN-γ+ T cells (d), CD8+ T cells (e), and CD8+/IFN-γ+ T cells (f) in cell suspensions prepared from the lungs isolated from Fas (−), FasL (−), and WT (C57BL/6) mice at 3, 7, 10, and 14 days of ECTV infection and from control, uninfected mice. The bars represent the mean from 5 separate experiments ± SEM.  *Significant differences with P ≤ 0.05 and ** P ≤ 0.01 in comparison to Fas- and FasL-deficient mice.
Figure 4
Figure 4
Lack of Fas or FasL during mousepox infection results in disturbances of cytokine and chemokine production. Relative mRNA levels of CXCL1, CXCL9, IL-10, IL-15, and TGF-β1 in the lungs isolated from Fas (−), FasL (−), and WT (C57BL/6) mice at 3, 7, 10, and 14 days of ECTV infection and from control, uninfected mice. The mRNA expressions were normalized by that of GAPDH, and the lungs of control, uninfected mice were estimated as 1. The bars represent the mean from 3 separate experiments ± SEM.  *Significant differences with P ≤ 0.05 and ** P ≤ 0.01.
Figure 5
Figure 5
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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