Fas Controls Neutrophil Lifespan during Bacterial and Viral Infection

Introduction

The regulation of neutrophil lifespan is critical for a circumscribed immune response. Neutrophils are sensitive to Fas/CD95 death receptor signaling in vitro but it is unknown if Fas regulates neutrophil lifespan in vivo. We hypothesized that FasL-expressing CD8⁺ T cells, which kill antigen-stimulated T cells during chronic lymphocytic choriomeningitis virus (LCMV) infection, can also induce neutrophil death in tissues during infection. Infection of Fas- and FasL-deficient mice with the enteropathogenic-like mouse pathogen Citrobacter rodentium (C.rodentium) is associated with neutrophil infiltration and severe diarrhoea. We hypothesized that a deficiency of Fas in neutrophils prolongs neutrophil lifespan and contributes to the accumulation of neutrophils in C.rodentium-infected mice.

Methods

Fas signaling can drive IL-1β and IL-18 production and thereby affect neutrophil accumulation in tissues, so mixed bone marrow chimeras were generated to compare the accumulation of WT and Fas-deficient neutrophils in vivo. For mixed bone-marrow chimeras, irradiated Ly5.1 mice were reconstituted with 2.5 x 10⁶ bone marrow cells from ubiquitin-GFP or Ly5.1/5.2 mice combined with LysM-Cre Fas^fl/fl at a 1:1 ratio. For infection studies, mice were infected with LCMV docile or C.rodentium. For in vitro assays, neutrophils were primed for 1 h with GM-CSF prior to treatment with Toll-like receptor (TLR) ligands or IL-18 and FcFasL. Neutrophil viability was measured by live cell imaging and automated image analysis.

Results

Using LysM-Cre Fas^fl/fl mice, which lack Fas expression in macrophages and neutrophils, we show that Fas regulates neutrophil lifespan in the colon during C.rodentium infection, and during LCMV infection in the lung, peripheral blood and spleen. To examine if pathogen-derived molecules can modulate Fas signaling in neutrophils, we primed neutrophils with TLR ligands, which ablated Caspase-8 processing and Fas signaling. Treatment of neutrophils with the TLR ligands Pam₃CSK₄, Pam₂CSK₄ or LPS or the MyD88-dependent cytokine IL-18 strongly protected neutrophils against Fas-induced death.

Conclusion

These data provide the first in vivo genetic evidence that neutrophil lifespan is controlled by death receptor signaling and provides a mechanism to account for neutrophil resistance to Fas stimulation during infection.