Lymphotoxin, but not TNF, is required for prion invasion of lymph nodes

Abstract

Neuroinvasion and subsequent destruction of the central nervous system by prions are typically preceded by a colonization phase in lymphoid organs. An important compartment harboring prions in lymphoid tissue is the follicular dendritic cell (FDC), which requires both tumor necrosis factor receptor 1 (TNFR1) and lymphotoxin β receptor (LTβR) signaling for maintenance. However, prions are still detected in TNFR1⁻/⁻ lymph nodes despite the absence of mature FDCs. Here we show that TNFR1-independent prion accumulation in lymph nodes depends on LTβR signaling. Loss of LTβR signaling, but not of TNFR1, was concurrent with the dedifferentiation of high endothelial venules (HEVs) required for lymphocyte entry into lymph nodes. Using luminescent conjugated polymers for histochemical PrP(Sc) detection, we identified PrP(Sc) deposits associated with HEVs in TNFR1⁻/⁻ lymph nodes. Hence, prions may enter lymph nodes by HEVs and accumulate or replicate in the absence of mature FDCs.

Figure 1. Repeated LTβR-Ig administration chronically downregulates LTβR signaling and de-differentiates FDC networks in C57BL/6 and TNFR1^−/− spleens and lymph nodes.

Frozen sections from spleens (A–D) and mesenteric lymph nodes (E–H) of C57BL/6 (WT) Ig-treated (A & E), C57BL/6 (WT) LTβR-Ig-treated (C & G), TNFR1^−/− Ig-treated (B & F), or TNFR1^−/− LTβR-Ig-treated (D & H) mice were analyzed by immunohistochemistry and developed with alkaline phosphatase for follicular dendritic cell marker 1 (FDCM1). The total number of FDCM1-positive (FDCM1+; black) or FDCM1-negative (FDCM1−; white) lymphoid follicles were scored for spleens (I) and mesenteric lymph nodes (mLNs; J) and expressed as a percentage of total follicles in each treatment group. FDCM1+ FDC networks were visible in 82% of WT Ig-treated spleen follicles (A; I) and 94% of WT-Ig lymph node follicles (E; J), whereas FDCM1+ FDCs were absent (0% of follicles) in spleens and lymph nodes from mice lacking TNFR1 and/or LTβR signaling (B–D; F–H; I–J). Total mRNA was isolated from spleens of mice from the indicated treatment groups and analyzed for expression of LTβR signaling targets CXCL13 (Mean ± S.E.M.: WT-Ig = 102.68±11.56, WT-LTβR-Ig = 18.05±2.30, TNFR1^−/−-Ig = 19.56±1.63, and TNFR1^−/−-LTβR-Ig = 15.14±0.15) (K) and NFκB2 (Mean ± S.E.M.: WT-Ig = 103.10±14.65, WT-LTβR-Ig = 33.30±2.10, TNFR1^−/−-Ig = 46.04±7.53, and TNFR1^−/−-LTβR-Ig = 33.39±1.18) (L) by Real Time PCR. Both CXCL13 and NFκB2 mRNA levels were reduced in all treatment groups relative to WT-Ig.

Figure 2. PrP^Sc accumulation in TNFR1^−/− lymph nodes requires LTβR signaling independent of Prnp expression.

C57BL/6 (WT) or TNFR1^−/− mice inoculated i.p. with 6 log LD₅₀ RML6 and treated weekly with control Ig or LTβR-Ig were sacrificed at 60 d.p.i. Histoblots were performed on frozen sections from spleens (SPL; A–D) or mesenteric lymph nodes (mLN; E–H) from mice in each treatment group to visualize PrP^Sc deposition. Whole organs are shown in left panels, and corresponding higher resolution images for each treatment group are shown in right panels. Note that lack of TNFR1 signaling can prevent PrP^Sc accumulation in spleen (B) but not lymph node (F). However, blocking LTβR signaling can prevent PrP^Sc accumulation in TNFR1^−/− lymph nodes (compare F and H). Prion infectivity titers in mLN homogenates from individual TNFR1^−/− Ig-treated and LTβR-Ig treated mice were measured using the scrapie cell assay (I). Whereas TNFR1^−/−-Ig mLNs all harbored ≥6.1 log TCI units/g tissue, prion infectivity in TNFR1^−/−-LTβR-Ig mLNs was reduced by at least 2.5 log TCI units/g tissue. Total mRNA was isolated from spleens (J) or mesenteric lymph nodes (mLN; K) of mice from the indicated treatment groups and analyzed for Prnp expression by Real Time PCR (Mean ± S.E.M.: Spleen – WT-Ig = 100.69±6.74, WT-LTβR-Ig = 46.02±4.30, TNFR1^−/−-Ig = 48.94±0.55, and TNFR1^−/−-LTβR-Ig = 50.11±0.55; mLN – WT-Ig = 106.05±22.54, WT-LTβR-Ig = 101.69±12.88, TNFR1^−/−-Ig = 56.43±3.13, and TNFR1^−/−-LTβR-Ig = 72.35±6.65). Whereas spleens from WT-LTβR-Ig, TNFR1^−/−-Ig, and TNFR1^−/−-LTβR-Ig mice all showed decreases in Prnp expression relative to WT-Ig spleens (J), no differences could be found in Prnp expression in mLNs from mice in any treatment group (K).

Figure 3. MadCam1 immunoreactivity in lymphoid tissue correlates with prion deposition.

Formalin-fixed cryosections from spleens (SPL; A–D) or mesenteric lymph nodes (mLN; F–I) of C57BL/6 (WT) Ig-treated (A & F), TNFR1^−/− Ig-treated (B & G), C57BL/6 (WT) LTβR-Ig-treated (C & H), or TNFR1^−/− LTβR-Ig-treated (D & I) mice were immunostained with an antibody against the stromal cell marker, mucosal addressin cell adhesion molecule 1 (MadCam1), and visualized with alkaline phosphatase. (E) The total number of MadCam1-positive (MadCam1+; black) or MadCam1-negative (MadCam1-; white) lymphoid follicles were scored for spleens and expressed as a percentage of total follicles in each treatment group. (J) The total number of MadCam1-postive (MadCam1+) structures per mesenteric lymph node (mLN) was counted and averaged for each treatment group. WT-Ig mLNs contained 47.5±14 MadCam1+ structures, WT-LTβR-Ig = 9.5±3, TNFR1^−/−-Ig = 74±19, and TNFR1^−/−-LTβR-Ig = 0. MadCam1 immunoreactivity in WT-Ig spleens was localized to the marginal sinus (A; black arrow) and 55% (E) of germinal centers (A; white arrow). This staining pattern was absent (0%; E) in the spleens of mice from all other treatment groups (B,C & D). In contrast, MadCam1 immunoreactivity in WT-Ig mLNs was largely found in thick vessels (F). MadCam1 immunoreactivity was retained in TNFR1^−/−-Ig mLNs (G) but absent in mLNs from mice treated with LTβR-Ig (H–J). Size bars: Left panels = 200 µm; Right panels = 100 µm.

Figure 4. Vessel-associated MadCam1 expression in lymph nodes is preserved in the absence of TNFR1 signaling.

Frozen sections from mesenteric lymph nodes (mLN) of C57BL/6 (WT) Ig-treated (A), TNFR1^−/− Ig-treated (B), C57BL/6 (WT) LTβR-Ig-treated (C), or TNFR1^−/− LTβR-Ig-treated (D) mice were analyzed by immunofluorescence with MadCam1 antibody. In WT-Ig mLNs (A), MadCam1 robustly stained thick vessels (yellow arrow), while germinal centers were weakly MadCam1-positive (white arrow). In TNFR1^−/−-Ig mLNs (B), MadCam1-positive germinal centers were absent, while vessel-associated MadCam1 staining persisted. In contrast, both WT-LTβR-Ig and TNFR1^−/−-LTβR-Ig mLNs were MadCam1-negative (C & D). Size bars = 100 µm. Total mRNA was isolated from spleens (E) or mesenteric lymph nodes (F) of C57BL/6 (WT) Ig-treated, C57BL/6 (WT) LTβR-Ig-treated, TNFR1^−/− Ig-treated, or TNFR1^−/− LTβR-Ig-treated mice and analyzed for MadCam1 expression by Real Time PCR (Mean ± S.E.M.: Spleen – WT-Ig = 104.21±16.56, WT-LTβR-Ig = 29.98±1.28, TNFR1^−/−-Ig = 30.41±2.56, and TNFR1^−/−-LTβR-Ig = 27.95±3.77; mLN – WT-Ig = 100.89±8.06, WT-LTβR-Ig = 45.40±6.75, TNFR1^−/−-Ig = 49.42±0.63, and TNFR1^−/−-LTβR-Ig = 24.79±7.25). MadCam1 expression was reduced in spleens of WT-LTβR-Ig-treated, TNFR1^−/− Ig-treated, and TNFR1^−/− LTβR-Ig-treated mice compared to WT-Ig. MadCam1 expression in WT-LTβR-Ig and TNFR1^−/− Ig mLNs was reduced compared to WT-Ig, and MadCam1 expression in TNFR1^−/− LTβR-Ig mLNs was reduced compared to TNFR1^−/−-Ig.

Figure 5. MadCam1-positive vessels in mesenteric lymph nodes are prion protein-expressing high endothelial venules.

Co-immunofluorescent confocal microscopy was performed on frozen sections from WT-Ig mesenteric lymph nodes with the high endothelial venule (HEV)-specific marker, peripheral node addressin (PNAd; green; A & D) and MadCam1 (red; B & E). Merged image shown in C & F. MadCam1-positive vessels co-localized with PNAd (A,B & C; yellow arrow). Note that MadCam1-positive germinal centers were PNAd-negative (B & C; white arrow). Standard co-immunofluorescence was performed on frozen sections from WT-Ig mesenteric lymph nodes with MadCam1 (red; G) and an antibody directed against the C-terminus of the prion protein (PrP; green; H; POM1; [62]). MadCam1-positive HEVs were also PrP-positive (I). Size bars in A–C = 150 µm. Size bars in D–F = 50 µm. Size bars in G–I = 20 µm.

Figure 6. PrP^Sc is present both in and around MadCam1-positive HEVs in TNFR1^−/−-Ig mesenteric lymph nodes.

TNFR1^−/− mice inoculated i.p. with 6 log LD₅₀ RML6 and treated weekly with control Ig were sacrificed at 60 d.p.i. Immunofluorescence (A–I) and histoblots (J & K) were then performed on frozen sections from prion-infected TNFR1^−/−-Ig mesenteric lymph nodes. Co-IF with anti-serum (XN) against PrP (green; A) and MadCam1 (red; B) showed points of intense PrP immunoreactivity localized to HEVs (C). Confocal co-IF with the amyloid-binding dye, p-FTAA (green; D) and MadCam1 (red; E,F) revealed some points of PrP^Sc association with HEVs (F); however much of the PrP^Sc was present outside of HEVs (I). Histoblots pre-stained with PNAd antibody and developed with AP (pink; J) also revealed some prion-infected HEVs (black arrows), some non-infected HEVs (white arrow), and some PrP^Sc deposits that were not HEV-associated (yellow arrow). (K) Total numbers of PNAd-positive HEVs in histoblot co-stains were counted and scored as PrP^Sc-positive (PrP^Sc+; black) or PrP^Sc-negative (PrP^Sc+; white), and total PrP^Sc deposits were counted and scored as PNAd-positive (PNAd+; black) or PNAd-negative (PNAd; white). 35% of HEVs were PrP^Sc-positive, and 58% of PrP^Sc deposits were PNAd-positive. Size bars in A–F = 50 µm. Size bars in G–I = 100 µm.