The Protein Kinase Double-Stranded RNA-Dependent (PKR) Enhances Protection against Disease Cause by a Non-Viral Pathogen

Abstract

PKR is well characterized for its function in antiviral immunity. Using Toxoplasma gondii, we examined if PKR promotes resistance to disease caused by a non-viral pathogen. PKR ^−/− mice infected with T. gondii exhibited higher parasite load and worsened histopathology in the eye and brain compared to wild-type controls. Susceptibility to toxoplasmosis was not due to defective expression of IFN-γ, TNF-α, NOS2 or IL-6 in the retina and brain, differences in IL-10 expression in these organs or to impaired induction of T. gondii-reactive T cells. While macrophages/microglia with defective PKR signaling exhibited unimpaired anti- T. gondii activity in response to IFN-γ/TNF-α, these cells were unable to kill the parasite in response to CD40 stimulation. The TRAF6 binding site of CD40, but not the TRAF2,3 binding sites, was required for PKR phosphorylation in response to CD40 ligation in macrophages. TRAF6 co-immunoprecipitated with PKR upon CD40 ligation. TRAF6-PKR interaction appeared to be indirect, since TRAF6 co-immunoprecipitated with TRAF2 and TRAF2 co-immunoprecipitated with PKR, and deficiency of TRAF2 inhibited TRAF6-PKR co-immunoprecipitation as well as PKR phosphorylation induced by CD40 ligation. PKR was required for stimulation of autophagy, accumulation the autophagy molecule LC3 around the parasite, vacuole-lysosomal fusion and killing of T. gondii in CD40-activated macrophages and microglia. Thus, our findings identified PKR as a mediator of anti-microbial activity and promoter of protection against disease caused by a non-viral pathogen, revealed that PKR is activated by CD40 via TRAF6 and TRAF2, and positioned PKR as a link between CD40-TRAF signaling and stimulation of the autophagy pathway.

Author Summary

PKR was identified more than 30 years ago as an inhibitor of viral replication. It is unknown if PKR promotes protection against disease caused by non-viral pathogens. We addressed this question using Toxoplasma gondii, a major parasitic pathogen. T. gondii can cause cerebral and/or eye disease primarily in immunosuppressed patients and newborns. After infection with T. gondii, PKR-deficient mice exhibited high parasite loads in the eye and brain and were more susceptible to ocular and cerebral toxoplasmosis. Macrophages and microglia are important effectors of protection against T. gondii. These cells required PKR signaling to kill the parasite in response to stimulation via CD40, a molecule that promotes protection against ocular and cerebral toxoplasmosis. CD40 functioned only through its TRAF6 binding site to activate PKR, but this process was also dependent on TRAF2 where this molecule likely acted as an intermediary that promoted TRAF6-PKR association and PKR activation. PKR linked CD40-TRAF signaling to stimulation of the autophagy pathway and T. gondii killing. Our studies identified a previously unappreciated role of PKR as mediator of anti-microbial activity and promoter of resistance against disease caused by a non-viral pathogen, as well as provided new insight on the molecular link between CD40 and PKR.