Early cytokine elevation, PrPres deposition, and gliosis in mouse scrapie: no effect on disease by deletion of cytokine genes IL-12p40 and IL-12p35

Abstract

Neurodegenerative diseases are typically associated with an activation of glia and an increased level of cytokines. In our previous studies of prion disease, the cytokine response in the brains of clinically sick scrapie-infected mice was restricted to a small group of cytokines, of which IL-12p40, CCL2, and CXCL10 were present at the highest levels. The goal of our current research was to determine the relationship between cytokine responses, gliosis, and neuropathology during prion disease. Here, in time course studies of C57BL/10 mice intracerebrally inoculated with 22L scrapie, abnormal protease-resistant prion protein (PrPres), astrogliosis, and microgliosis were first detected at 40 days after intracerebral scrapie inoculation. In cytokine studies, IL-12p40 was first elevated by 60 days; CCL3, IL-1β, and CXCL1 were elevated by 80 days; and CCL2 and CCL5 were elevated by 115 days. IL-12p40 showed the most extensive increase throughout disease and was 30-fold above control levels at the terminal stage. Because of the early onset and dramatic elevation of IL-12p40 during scrapie, we investigated whether IL-12p40 contributed to the development of prion disease neuropathogenesis by using three different scrapie strains (22L, RML, 79A) to infect knockout mice in which the gene encoding IL-12p40 was deleted. We also studied knockout mice lacking IL-12p35, which combines with IL-12p40 to form active IL-12 heterodimers. In all instances, knockout mice did not differ from control mice in survival time, clinical tempo, or levels of spongiosis, gliosis, or PrPres in the brain. Thus, neither IL-12p40 nor IL-12p35 molecules were required for prion disease-associated neurodegeneration or neuroinflammation.

Fig 1

Early detection of PrPres in scrapie-infected brains. (A) Immunoblot detection of PrPres in brain at various times after i.c. inoculation with scrapie strain 22L. The upper panel was exposed for 10 s, and the lower panel was exposed for 30 min to increase sensitivity. Exposure for 120 min did not reveal any bands in the 20-dpi samples. Results similar to those in the lower panel were also seen when samples were analyzed using the phosphotungstic acid precipitation method (28). (B) PrPres was quantitated by density scanning of the gels shown in panel A. The average amount of PrPres detected at each time point is shown as a percentage of the amount detected at the terminal stage.

Fig 2

Early detection of PrPres and microglia by immunohistochemistry following scrapie infection. (A to E) Staining with the monoclonal anti-PrP antibody D13 for detection of PrPres (brown) in the thalamus and forebrain at various days postinoculation. (F to -J) Staining of microglia with anti-Iba1 in brain sections parallel to those studied in panels A to E. (A) At 20 dpi, no PrPres was detected; however, weak background staining of PrPsen was seen. Similar staining was seen in uninfected mice but was not seen in PrP-null mice, which confirmed its identity as PrPsen. (B) At 40 dpi, two patchy areas of PrPres were detected, one in the thalamus on the right and several small groups of staining in the forebrain on the left. (C and D) Enlargements of panel B. (E) At 80 dpi, PrPres was found over a more extensive area. (F) Anti-Iba1 staining at 20 dpi detected an even distribution of microglia in the same area. (Inset) Thin processes of cells characteristic of nonactivated microglia. (G) At 40 dpi, increased staining of Iba1 was seen near the same areas where PrPres was found (compare panel B). (H and I) Microglia with short thick processes and enlarged somata typical of activated microglia. (J) At 80 dpi, microglia with larger cell bodies were seen over most of the field shown, and the inset confirmed the morphology of activated cells. Bars, 250 μm (A, B, E, F, G, J), 100 μm (C and D), and 50 μm (H, I, and insets to panels F and J).

Fig 3

Detection of cytokines in brain tissue at various times after scrapie infection. Protein levels of 9 cytokines in the brains of scrapie 22L-infected mice were measured by multiplex assays at various days postinoculation. The six cytokines for which results are shown had values significantly elevated over those for normal control mice (N) at various days postinoculation. Data for CCL2 were obtained from samples assayed at a 4% brain concentration, and other cytokines were assayed at a 1% brain concentration. IL-9, IL-13, and gamma interferon were also analyzed, but no values above those for normal controls were detected. Two to four mice per group were studied. Statistical analysis was carried out using one-way ANOVA with GraphPad Prism software. ***, P < 0.001; **, P < 0.01; *, P < 0.05.

Fig 4

Scrapie incubation times and PrPres detection in infected knockout mice. (A and B) The periods of incubation until clinical disease in scrapie-infected IL-12p40 KO (A) and IL-12p35 KO (B) mice were compared to those in non-knockout B6 controls. Mice were inoculated intracerebrally with scrapie strain 22L, RML, or 79A. Because experiments were carried out at separate times, each KO mouse strain had a separate set of B6 controls, which were inoculated with the same dilution of the three scrapie strains. Each dot represents one mouse. Horizontal bars show the mean incubation period. Values represent the day postinoculation when the animal was euthanized due to advanced signs of clinical scrapie as described in Materials and Methods. (C and D) Immunoblot detection of PrPres in the brains of representative IL-12p40 KO (C) and IL-12p35 KO (D) mice and their respective control B6 mice at the clinical endpoint after infection by scrapie strain 22L, RML, or 79A. All samples were treated with proteinase K as described in Materials and Methods. Each lane was loaded with 0.5-mg tissue equivalents. The blot was probed using anti-PrP antibody D13 and was developed using enhanced chemiluminescence detection. Blots C and D were exposed for 2.5 and 1.0 min, respectively.

Fig 5

Detection of microgliosis, astrogliosis, and vacuolation in scrapie-infected knockout and control B6 mice. Mice were euthanized at the clinical endpoint (approximately 135 dpi) after infection with scrapie strain 22L. Control mice of each strain were inoculated with normal brain homogenate (NBH) and were euthanized at the same time point. (A and B) Immunohistochemical analysis using anti-Iba1 (A) or anti-GFAP (B) shows that the density and morphology of activated microglia and astroglia in infected mice were similar and differed from those of normal glia seen in NBH-infected controls. (C) H&E staining shows vacuolation of gray matter in all infected mice but not in NBH-infected controls. Images are from the thalamus, but similar observations were made in other gray-matter regions. Bars, 200 μm. All panels within each subgroup are at the same magnification.