A lympho-follicular microenvironment is required for pathological prion protein deposition in chronically inflamed tissues from scrapie-affected sheep.

In sheep scrapie, pathological prion protein (PrP(Sc)) deposition occurs in the lymphoreticular and central nervous systems. We investigated PrP(Sc) distribution in scrapie-affected sheep showing simultaneous evidence of chronic lymphofollicular, lymphoproliferative/non-lymphofollicular, and/or granulomatous inflammations in their mammary gland, lung, and ileum. To do this, PrP(Sc) detection was carried out via immunohistochemistry and Western Blotting techniques, as well as through inflammatory cell immunophenotyping. Expression studies of gene coding for biological factors modulating the host's inflammatory response were also carried out. We demonstrated that ectopic PrP(Sc) deposition occurs exclusively in the context of lymphofollicular inflammatory sites, inside newly formed and well-organized lymphoid follicles harboring follicular dendritic cells. On the contrary, no PrP(Sc) deposition was detected in granulomas, even when they were closely located to newly formed lymphoid follicles. A significantly more consistent expression of lymphotoxin α and β mRNA was detected in lymphofollicular inflammation compared to the other two types, with lymphotoxin α and β signaling new lymphoid follicles' formation and, likely, the occurrence of ectopic PrP(Sc) deposition inside them. Our findings suggest that, in sheep co-affected by scrapie and chronic inflammatory conditions, only newly formed lymphoid follicles provide a suitable micro-environment that supports the scrapie agent's replication in inflammatory sites, with an increased risk of prion shedding through body secretions/excretions

qRT-PCR expression of PrP gene in sheep mammary glands.

<p>Transcript levels are shown in mammary glands with lymphofollicular, lymphoproliferative/non-lymphofollicular, and granulomatous mastitis, as well as in histologically and microbiologically healthy ovine mammary glands (Groups A, B, C, and D, respectively, as described in “Materials and Methods”). Transcripts were normalized to housekeeping sheep 18S gene level. Differences in PrP gene expression levels were calculated as fold changes in transcripts between the mammary gland tissue samples under study <i>versus</i> one histologically and microbiologically healthy sheep mammary gland, which was used as the reference value. The box represents the 25^th to 75^th quartile, the whiskers represent the range, and the horizontal line in the box is the median. White dots beyond the whiskers indicate outliers. The sample values were normalized to housekeeping sheep 18S gene level.</p

PrP quantification in ovine mammary glands.

<p>PrP is quantified in mammary glands with lymphofollicular, lymphoproliferative/non-lymphofollicular, and granulomatous mastitis, as well as in histologically and microbiologically healthy sheep mammary glands (Groups A, B, C, and D, respectively, as described in “Materials and Methods”). Differences among the 4 groups are represented as PrP optical density (OD) signals. The mammary gland samples were normalized in relation to the total protein amount.</p

Inflammatory lesions patterns in ovine mammary glands.

<p>Micrographs show the inflammatory lesions’ morphology in mammary glands from sheep concurrently affected by scrapie and mastitis. Representative patterns of lymphofollicular (A), granulomatous (B), and lymphoproliferative/non-lymphofollicular (C) mastitis. A histologically normal ovine mammary gland is also shown (D). Hematoxylin-eosin (H&E) stain. Scale bar = 100 µm.</p

Cytokine, chemokine, receptor, adhesion molecule, and PrP gene expression levels.

<p>For the different groups the data are shown as mean values ± SD. Multiple comparisons were made with Tukey’s test.</p>*<p>P<0.05 values were considered to be statistically significant.</p><p>L =  lymphofollicular; LnL =  lymphoproliferative/non-lymphofollicular; G =  granulomatous; H =  healthy.</p

Differential qRT-PCR gene expression for cytokines, chemokines, receptors, and adhesion molecules involved in inflammatory responses.

<p>Total mRNA was isolated from inflamed sheep mammary glands showing lymphofollicular, lymphoproliferative/non-lymphofollicular, and granulomatous mastitis, as well as from histologically and microbiologically healthy ovine mammary glands (Groups A, B, C, and D, respectively, as described in “Materials and Methods”). Differences in specific gene expression levels were calculated as fold changes in transcripts between the mammary gland tissue samples under study <i>versus</i> one histologically and microbiologically healthy sheep mammary gland, which was used as the reference value. Each boxplot represents log-transformed gene expression levels in the different groups of cytokines, chemokines, and their receptors, together with adhesion molecules. The box represents the 25^th to 75^th quartile, the whiskers represent the range, and the horizontal line in the box is the median. White dots beyond the whiskers indicate outliers. The sample values were normalized to housekeeping sheep 18S gene level. The X-axis legend is common for all 14 graphics. <i>P</i> values among the different groups are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062830#pone-0062830-t003" target="_blank">Table 3</a>.</p

Western Blotting (WB) analysis for PrP^Sc in ovine mammary glands with chronic mastitis.

<p>Lines: 1–2 =  brain from a naturally scrapie-affected (Scr⁺) or scrapie-free (Scr^-) sheep. Lines: 3–4–8 =  mammary glands from scrapie-affected sheep displaying lymphofollicular mastitis. Lines: 5–6–7 =  mammary glands from scrapie-affected sheep displaying granulomatous mastitis. Line 9 =  mammary gland from a scrapie-affected sheep displaying lymphoproliferative/non-lymphofollicular mastitis. Line 10 =  palatine tonsil from a naturally scrapie-affected sheep showing PrP^Sc in mammary gland with lymphofollicular mastitis. Among the mammary glands, positive WB signals were observed only in the case of lymphofollicular mastitis.</p

PrP^Sc is detected only in inflamed sheep mammary glands containing newly formed lymphoid follicles.

<p>Inflammatory cell immunophenotyping in serial paraffin-embedded sections of mammary glands from sheep simultaneously affected by scrapie and lymphofollicular, lymphoproliferative/non-lymphofollicular, or granulomatous mastitis. A normal ovine palatine tonsil is also shown for comparison. CNA-42, CD3, and CD79 were used as markers for FDCs, T cells, and B cells (Panel A), while CD172a, CD68, and CD11c were used for activated macrophages and DCs (Panel B), respectively. In panel C, a representative set of negative controls are shown. These were obtained by omitting all the primary antibodies used on the tonsil sections (photos 1, 2, 3, 4, 6, and 7), as well as from granulomatous mastitis (photo 5). The omitted primary antibodies are indicated on each photo. It is noteworthy that the relative positioning of these cell populations was similar in newly formed lymphoid follicles and constitutive follicles normally hosted inside the palatine tonsil. CD172a⁺ macrophages were the most representative cell population in granulomatous mastitis, which also displayed a moderate presence of T and B cells and DCs. CD3⁺ T cells were the most abundant cellular component in the context of lymphoproliferative/non-lymphofollicular mastitis. PrP^Sc deposition occurred in co-localization with CD79⁺ B cells and the CNA42⁺ FDC network both in palatine tonsil and in newly formed lymphoid follicles. Immune reactions were visualized by 3-3′-diaminobenzidine (DAB) chromogen, with F99 primary MoAb being used for PrP^Sc detection. Mayer’s hematoxylin counterstain. Scale bar = 100 µm.</p

Inflammatory lesion patterns and PrP^Sc deposition in ovine lungs.

<p>Micrographs show the inflammatory lesions’ morphology and PrP^Sc deposition in lungs from sheep simultaneously affected by scrapie and parasitic bronchopneumonia caused by nematodes. Representative patterns of lymphofollicular (A) and lymphofollicular (arrows) associated with granulomatous (arrowheads) inflammation (B and D). A histologically normal ovine lung is also shown (C). PrP^Sc deposits (arrows) are visible within newly formed lymphoid follicles adjacent to granulomatous lesions (E and F). Micrograph F is a higher magnification of the red line-enclosed area shown in micrograph E. Hematoxylin-eosin (H&E) stain (A, B, C, and D); PrP^Sc immunohistochemistry (IHC) with F99 as primary antibody and Mayer’s hematoxylin counterstain (E and F). Scale bar = 100 µm.</p

Details of the primers used to determine cytokine, chemokine, and adhesion molecule gene expression levels.

*<p>Sheep 18S gene was used as housekeeping control gene.</p