Figure 1.
Cre-mediated gene recombination in FDC in the spleens, lymph nodes and Peyer's patches of CD21-Cre ROSA26flox/Flox mice.
A) Analysis of the cellular sites of LacZ expression (blue) in the spleens, inguinal lymph nodes, Peyer's patches and mesenteric lymph nodes of CD21-Cre ROSA26flox/flox mice shows Cre-mediated recombination in a focus of cells within the B cell follicles. Sections were counterstained with nuclear fast red (red). B) IHC analysis of FDC (CD35+ cells, upper row, red) and B cells (CD45R+ cells, lower row, red) confirmed that Cre-mediated LacZ expression (blue) was associated with FDC in the spleens of WT→CD21-Cre ROSA26flox/flox mice. No LacZ expression was associated with FDC in spleens from ROSA26flox/flox mice that lacked Cre. C) IHC analysis of the status of FDC (CD35+ and C4-binding cells; red) and B cells expressing CD45R, CD19, and CD1d (red) in spleens from WT, CD21-Cre ROSA26flox/flox, WT→CD21-Cre ROSA26flox/flox and ROSA26flox/flox mice. Scale bars 100 µm. n = 6 mice/group.
Figure 2.
FDC-restricted PrPc expression in the spleens of Prnpstop/-→CD21-Cre Prnpstop/- mice.
A) The anticipated distribution of PrPC expression on FDC and B cells in tissues from each mouse group. B) The detection of Cre in the tail and spleen but not blood of the Prnpstop/-→CD21-Cre Prnpstop/- mice confirmed the restriction of the Cre-expression to the stromal but not haematopoietic compartments of these mice (upper panel). Efficient Cre-mediated recombination of Prnpstop (Prnpstop(R)) was restricted to the FDC-containing stromal compartment of the spleens of Prnpstop/-→CD21-Cre Prnpstop/- mice when compared to control mice. Cre-mediated recombination by CD21-expressing lymphocytes was efficiently prevented in these mice by the irradiation and transfer of Prnpstop/- bone marrow as demonstrated by the lack of a Prnpstop(R) band in DNA extracted from blood (lower panel). B, blood; S, spleen; T, tail; M, DNA size markers; a, b, c, control DNA samples for each transgene combination tested which were (a) Prnp-/-, (b) Prnpstop/- and (c) complete recombination of the stop cassette within the Prnpstop/- allele. C) IHC analysis of PrPC expression (blue) by FDC (CD35+ cells; red) and sympathetic nerves (TH+ cells, green) confirmed PrPC expression was restricted to FDC in spleens of Prnpstop/-→CD21-Cre Prnpstop/- mice. Scale bar, 100 µm. D) Morphometric analysis confirmed that the magnitude of the PrPC expression co-localized upon the surfaces of FDC in the spleens of Prnpstop/-→CD21-Cre Prnpstop/- mice was similar to that observed upon FDC in spleens from Prnp+/-→Prnp+/- control mice (p<0.690, n = 48 FDC networks/group). In contrast, in the absence of Cre-recombinase expression by FDC in CD21-Cre Prnpstop/-→Prnpstop/- mice, PrPC expression was substantially lower than that observed upon FDC in spleens from Prnp+/-→Prnp+/- control mice (p<1×10-25, n = 48 FDC networks/group). E) Morphometric analysis confirmed that PrPC expression upon the surfaces of sympathetic nerves in the spleens of Prnpstop/-→CD21-Cre Prnpstop/-, CD21-Cre Prnpstop/-→ CD21-Cre Prnpstop/- and CD21-Cre Prnpstop/-→Prnpstop/- mice was significantly ablated when compared to that observed upon sympathetic nerves in spleens from Prnp+/-→Prnp+/- control mice (p<1×10-25, n = 48 sympathetic nerves/group). For all panels n = 6 mice/group.
Figure 3.
Effect of FDC-restricted PrPc expression on the spleens of Prnpstop/-→CD21-Cre Prnpstop/- mice.
A) IHC analysis of the status of FDC networks (C4-binding cells and CD21/CD35+ cells; red), B cells expressing CD45R (red), and CD3+ T cells (green). Morphometric analysis confirmed that there were no significant difference in the size (B) and number (C) of the CD35+ FDC networks in spleens from Prnpstop/-→CD21-Cre Prnpstop/-, CD21-Cre Prnpstop/-→CD21-Cre Prnpstop/-, CD21-Cre Prnpstop/-→Prnpstop/- mice and Prnp+/-→Prnp+/- control mice (n = 32 FDC networks/group). D and E), Comparison of the sympathetic innervation in spleens from each mouse group. D) IHC detection of TH-positive sympathetic nerves (green) and FDCs (CD35+ cells; red). Scale bar, 50 µm. E) Quantitative analysis of the relative positioning of the FDC and sympathetic nerves showed there were no significant differences in average distance between these cell populations in spleens from each mouse group (P = 0.932, n = 48 FDC networks/group). For all panels n = 6 mice/group.
Figure 4.
Effect of FDC-restricted PrPc expression on PrPSc accumulation in the spleen.
Mice were injected i.p. with the ME7 scrapie agent and tissues collected 35, 70 days and 105 days after exposure. A and B) High levels of PrPd were detected in association with FDC (CD21/35 positive cells) in the B cell follicles (CD45R positive cells) of spleens of mice with PrPC-expressing FDC: Prnpstop/-→CD21-Cre Prnpstop/- mice, CD21-Cre Prnpstop/-→CD21-Cre Prnpstop/- mice and Prnp+/-→Prnp+/- control mice. B) High magnification images of the sites of PrPd accumulation (red) at 70 days post-injection with scrapie. Arrowheads show PrP-accumulation within tingible body macrophages. C) Analysis of adjacent sections by PET-immunoblot analysis confirmed the presence of PK-resistant PrPSc (blue/black). In contrast, no PrPd or PrPSc was detected in spleens of CD21-Cre→Prnpstop/-mice that lacked PrPC-expressing FDC. Arrows indicate PrPSc accumulation upon FDC. A, scale bar = 100 µm. B, scale bar = 20 µm. C, scale bar = 500 µm. For all panels n = 4 mice/group.
Figure 5.
FDC-restricted PrPc-ablation in the spleens of Prnpflox/-→CD21-Cre Prnpflox/- mice.
A) The anticipated distribution of PrPC expression on FDC and B cells in tissues from each mouse group. B) PCR analysis of DNA isolated from the spleens, blood and tails of Prnpflox/-→CD21-Cre Prnpflox/- mice confirmed that efficient Cre-mediated DNA recombination and Prnp-ablation (Prnpdeflox) was restricted to the FDC-containing stromal compartment of the spleen. Cre-mediated recombination of CD21-expressing lymphocytes was efficiently prevented in these mice by the irradiation and transfer of Prnpflox/- bone marrow as demonstrated by the lack of a Prnpdeflox band in DNA extracted from blood (lower panel). B, blood; S, spleen; T, tail; M, DNA size markers; a, b, c, d control DNA samples for each transgene combination tested which were (a) Prnpflox/flox,(b) Prnpflox/-, (c) Prnp-/- and (d) Prnpflox/- with complete recombination of the floxed exon 3. C) IHC analysis of PrPC expression (blue) by FDC (CD35+ cells; red) and sympathetic nerves (TH+ cells, green) confirmed the PrPC-ablation was restricted to FDC in spleens of Prnpflox/-→CD21-Cre Prnpflox/- mice. Scale bar, 100 µm. D) Morphometric analysis confirmed that the magnitude of the PrPC expression co-localized upon the surfaces of FDC in the spleens of Prnpflox/-→CD21-Cre Prnpflox/- mice was significantly lower than that observed upon FDC from Prnp+/-→Prnp+/- control mice (P<1.0×10-24, n = 48 FDC networks/group). In contrast, in the absence of Cre-recombinase expression by FDC in CD21-Cre Prnpflox/-→Prnpflox/-mice, PrPC expression was similar to that observed upon FDC in spleens from Prnp+/-→Prnp+/- control mice (P = 0.106, n = 48 FDC networks/group). E) Morphometric analysis confirmed that the magnitude of the PrPC expression co-localized upon the surfaces sympathetic nerves in the spleens of Prnpflox/-→CD21-Cre Prnpflox/-, CD21-Cre Prnpflox/-→ CD21-Cre Prnpflox/- and CD21-Cre Prnpflox/-→Prnpflox/- mice was not significantly different when compared to that observed upon sympathetic nerves in spleens from Prnp+/-→Prnp+/- control mice (p = 0.400, n = 48 sympathetic nerves/group). For all panels n = 6 mice/group.
Figure 6.
Effect of FDC-restricted PrPC-ablation on FDC status.
A) IHC analysis of the status of FDC (C4-binding cells and CD21/CD35+ cells; red), B cells expressing CD45R (red), and CD3+ T cells (green). Morphometric analysis confirmed that there were was no significant difference in the size (B) and number (C) of the CD35+ FDC networks in spleens of mice from each mouse group (n = 32 FDC networks/group). D and E), Comparison of the sympathetic innervation in spleens from Prnpflox/-→CD21-Cre Prnpflox/-, CD21-Cre Prnpflox/-→CD21-Cre Prnpflox/-, CD21-Cre Prnpflox/-→Prnpflox/-mice and Prnp+/-→Prnp+/- control mice. D) IHC detection of TH-positive sympathetic nerves (green) and FDCs (CD35+ cells; red). Scale bar, 50 µm. E) Quantitative analysis of the relative positioning of the FDC networks and sympathetic nerves showed there was no significant difference in the average distance between these cell populations in spleens from each mouse group (P = 0.765, n = 48 FDC networks/group). For all panels n = 6 mice/group.
Figure 7.
Effect of FDC-restricted PrPC-ablation on immune complex trapping.
A) Mice were passively immunized with preformed PAP immune complexes, and 24 h later, the presence of immune complexes (red) upon FDC (CD35+ cells, green) assessed by IHC. Scale bar, 100 µm. B) Morphometric analysis confirmed that the magnitude of the immune complex-trapping co-localized upon the surfaces of FDC from Prnp-ablated Prnpflox/-→CD21-Cre Prnpflox/- mice was not significantly different from that observed in spleens from control mice. This analysis also confirmed that the immune complexes were preferentially associated with FDC in these tissues and significantly greater than the null hypothesis that the pixels were randomly distributed. *, P<1×10−21; **, P<1×10−32; *** P<9×10−28; n = 40 FDC networks/group. For all panels n = 6 mice/group.
Figure 8.
Effect of FDC-restricted PrPC-ablation on PrPSc accumulation in the spleen.
Mice were injected i.p. with the ME7 scrapie agent and tissues collected 70 days after exposure. A) High levels of PrPd (red, left-hand column) were detected in association with FDC (red, middle column) in the B cell follicles (red, right-hand column) of spleens from CD21-Cre Prnpflox/-→Prnpflox/-mice and Prnp+/-→Prnp+/- control mice that contained PrPC-expressing FDC. B) High magnification images of the sites of PrPd accumulation (red) at 70 days post-injection with scrapie. C) PET blot analysis of analysis of adjacent sections by PET-immunoblot analysis confirmed presence of PK-resistant PrPSc (blue/black). In contrast, no PrPSc was detected in spleens of Prnpflox/-→CD21-Cre Prnpflox/- and CD21-Cre Prnpflox/-→CD21-Cre Prnpflox/- mice that lacked PrPC-expressing FDC. In the spleens of some of these mice, low levels of PrPd were occasionally localised within tingible body macrophages (B, arrowheads). A, scale bar = 100 µm. B, scale bar = 20 µm. C, scale bar = 500 µm. Arrows indicate PrPSc accumulation upon FDC. For all panels n = 4 mice/group.
Figure 9.
Effect of FDC-restricted PrPC-ablation on PrPSc accumulation in the brains and spleens of scrapie-affected mice.
Control mice (Prnp+/- mice) and Prnpflox/-→CD21-Cre Prnpflox/- mice that lacked PrPC-expressing FDC were injected i.c. with the scrapie agent directly into the CNS. Brains and spleens were collected from clinically scrapie-affected mice to compare the neuropathology and cellular sites of PrPSc accumulation. A) High levels of spongiform pathology (H&E, upper row), heavy accumulations of PrPd (brown, second row), reactive astrocytes expressing GFAP (brown, third row) and active microglia expressing Iba-1 (brown, bottom row) were detected in the hippocampi of the brains of all clinically scrapie-affected mice. Scale bars, 500 µm. B) High levels of PrPd (red) were detected in association with FDC in spleens from clinically scrapie-affected control mice that contained PrPC-expressing FDC. C) PET blot analysis of analysis of adjacent sections by PET-immunoblot analysis confirmed the presence of PK-resistant PrPSc (blue/black). In contrast, no PrPd or PrPSc was detected in spleens of Prnpflox/-→CD21-Cre Prnpflox/- that lacked PrPC-expressing FDC. Scale bars = 500 µm.
Table 1.
PCR primers used to confirm the genotypes of mice used in this study.