In the present experiments, we found evidence for PrPres deposition and both astrogliosis and microgliosis in the brain at 40 days after intracerebral scrapie infection. In contrast, at 20 dpi, none of these findings were detectable. Thus, these three aspects of prion disease pathology appeared nearly simultaneously between 20 and 40 dpi. PrPres detection at 40 dpi in this system was 20 to 30 days earlier than in several previous studies (1
). This might be due to the rapid tempo of the disease induced by our stock of strain 22L, which is known to induce clinical disease at 135 to 140 dpi and is about 15 days faster than our stocks of other scrapie strains, such as RML, ME7, and 79A. This early time of onset of the pathological process in the brain should provide an opportunity for future detection of key early events in scrapie pathogenesis.
Using quantitative multiplex cytokine protein analyses with the same group of mice, we found elevated levels of several cytokines, including IL-12p40, CCL2, CCL3, CCL5, IL-1β, and CXCL1, starting at 60 to 80 dpi. These cytokines were previously shown to be present in scrapie-infected brains at the end stage of disease and were also produced by microglia and/or astroglia after in vitro
stimulation with scrapie-infected brain extracts (35
). These cytokines appeared slightly later than the initial PrPres deposits and gliosis, suggesting that cytokine elevation might be induced by these earlier pathological events. Alternatively, our biochemical cytokine assay based on analysis of whole-brain homogenates might not be sensitive enough to detect small foci of early cytokine production simultaneous with, or even preceding, PrPres deposition and gliosis. Because of these caveats, we cannot distinguish whether these cytokines contribute to the ongoing prion infection-induced brain damage and/or to the host response attempting to repair this damage.
The study of mice in which individual cytokine genes have been deleted is a powerful tool for testing the requirement for individual genes in disease pathogenesis or recovery. In previous studies of cytokine or cytokine receptor knockout mice using i.c. scrapie infection, a variety of outcomes have been seen. Depending on the individual gene tested and the scrapie strain utilized, survival times either were unchanged (IL-6 [19
], IL-4 and/or IL-13 [33
], MyD88 [23
]), were slightly increased (CCL2 with strain ME7 [8
], CXCR3 [26
], or IL-1R [29
]), or were slightly decreased (CCL2 with strain RML/Chandler [22
], IL-10 [33
], or CCR1 [16
]). Thus, although most of these cytokines, cytokine receptors, and cytokine regulators would be expected to influence neuroinflammation and neurotoxicity after scrapie infection, no single cytokine or receptor has so far been proven to be required for scrapie disease.
In the current experiments, we attempted to determine whether prion disease brain pathogenesis was influenced by deletion of IL-12p40, the cytokine with the earliest onset and highest fold increase during disease. Knockout mice in which expression of IL-12p35 was deleted were also studied, because a heterodimer of IL-12p40 and IL-12p35 polypeptides forms the active IL-12 cytokine. In these experiments, scrapie was inoculated by the i.c. route in order to focus the study on pathogenesis within the central nervous system (CNS) rather than on aspects of extracerebral infection and neuroinvasion (14
). After scrapie infection, these two KO mouse strains showed no differences in survival time, PrPres occurrence, astrogliosis, microgliosis, or neutrophil vacuolation from non-KO C57BL/6 control mice ( and ). Thus, none of the three cytokines containing IL-12p40 or IL-12p35—i.e., IL-12, IL-23, and the IL-12p40 dimer (see reference 6
for a review)—were required for these aspects of scrapie pathogenesis, nor were they required for fatal scrapie disease. Since many of these cytokines act on T lymphocytes and dendritic cells not found in the CNS during scrapie disease, it may not be surprising that no effect on scrapie was seen by deletion of these genes. However, in microglia, IL-12p70, the IL-12p40 monomer, and the IL-12p40 dimer all stimulate mitogen-activated protein (MAP) kinase and extracellular signal-regulated kinase (ERK) pathways, as well as inducing the production of tumor necrosis factor (TNF) and nitric oxide (10
). Nevertheless, based on our current experiments, these effects on microglial function do not influence scrapie disease tempo or survival after inoculation of any of the three scrapie strains tested in our studies.