<i>Alox15b</i> knockdown in <i>LDLr^−/−</i> mice.

<p><b>A</b>) Immunohistochemical detection of macrophages and ALOX15B in <i>Ldlr^−/−</i> mice. <b>B</b>) Quantification of <i>Alox15b</i> expression, normalized to <i>Emr1</i> expression (macrophage marker) in aortic tissue using Q-PCR (n = 2 for control and n = 3 for <i>Alox15b</i> shRNA). The sections presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043142#pone-0043142-g002" target="_blank">figure 2A</a> were stained with Mayer's hematoxylin while the quantified sections used for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043142#pone-0043142-g002" target="_blank">figure 2B</a> were not. <b>C</b>) Western blotting of ALOX15B and MAC-2 (macrophage marker) in aortic tissue. <b>D</b>) Quantification of <i>Alox15b</i> expression in bone marrow macrophages (BMM) isolated and differentiated at the end of the silencing experiment using Q-PCR (n = 2 for control and n = 3 for <i>Alox15b</i> shRNA). <b>E</b>) Western blotting of ALOX15B and ACTB in bone marrow macrophages. <b>F</b>) ALOX15B levels measured by immunohistochemistry in sections from aortic sinus from control and <i>Alox15b</i> knockdown mice (n = 7 per group). Data are presented as mean±SEM.</p

Quantification of atherosclerotic lesion area in <i>en face</i> prepared aortas from male hSAA1 (n = 33) and wt (n = 23) mice on ApoE-deficient background.

<p>Lesion area with positive Sudan IV staining is expressed as the percentage of total area in (A) total aorta, (B) aortic arch, (C) thoracic aorta and (D) abdominal aorta. Data are presented as mean ± SEM. ns = non significant with Mann-Whitney U-test. (E) Photographs illustrating atherosclerotic lesions in wt and hSAA1 mice.</p

Antibodies used in this study.

1<p>Oxford Biochemical research,</p>2<p>BD Bioscience,</p>3<p>Nordic biosite,</p>4<p>Polyclonal,</p>5<p>Monoclonal,</p>6<p>Horse rabbit peroxidase,</p>7<p>GE healthcare.</p

Decreased atherosclerotic lesions in aortas in <i>Alox15b</i> knockdown mice.

<p><b>A</b>) Plasma cholesterol, <b>B</b>) plasma triglycerides and <b>C</b>) body weight of <i>Alox15b</i> knockdown and control mice. <b>D</b>) Representative photographs showing aorta pinned out by en face technique and stained with Sudan IV. <b>E</b>) Quantification of subendothelial lipid accumulation in the aorta (n = 7 per group). <b>F</b>) Representative histological analysis of the aortic sinus stained with Oil Red O. <b>G</b>) Quantification of subendothelial lipid accumulation in the aortic root (n = 6 per group). Data are presented as mean±SEM.</p

Analysis of plaque composition and plasma levels of IL-2.

<p>Sections from aortic sinuses were stained with antibodies against <b>A</b>) CD4/CD8 (T cells), <b>B</b>) MAC-2 (macrophages), <b>C</b>) α-actin (smooth muscle cells), and <b>D</b>) collagen. Data are presented as mean±SEM. <b>E</b>) Plasma levels of soluble IL-2. (n = 6 per group).</p

Decreased lipid uptake and immunological signaling in human <i>ALOX15B</i>-silenced macrophages.

<p>Lipid accumulation was analyzed in human primary macrophages transfected with nonsilencing control siRNA or <i>ALOX15B</i> siRNA using Oil Red O staining after incubation with DMOG. <b>A</b>) Quantification of <i>ALOX15B</i> expression normalized to <i>ActB</i> expression measured with Q-PCR. <b>B</b>) Quantification of <i>ALOX15A</i> expression normalized to <i>ActB</i> expression measured with Q-PCR. <b>C</b>) Representative picture showing Oil Red O staining of control and <i>ALOX15B</i>-silenced macrophages (Scale bar = 50 µm). <b>D</b>) Quantification of Oil Red O staining in human primary macrophages (n = 7) normalized to control. <b>E</b>) Size of control and <i>ALOX15B</i>-silenced human primary macrophages (foam cells). <b>F</b>) Quantification of secreted cytokines in media from human primary macrophages (n = 7). Data are presented as mean±SEM normalized to control.</p

DataSheet_1_Immune mapping of human tuberculosis and sarcoidosis lung granulomas.docx

Tuberculosis (TB) and sarcoidosis are both granulomatous diseases. Here, we compared the immunological microenvironments of granulomas from TB and sarcoidosis patients using in situ sequencing (ISS) transcriptomic analysis and multiplexed immunolabeling of tissue sections. TB lesions consisted of large necrotic and cellular granulomas, whereas “multifocal” granulomas with macrophages or epitheloid cell core and a T-cell rim were observed in sarcoidosis samples. The necrotic core in TB lesions was surrounded by macrophages and encircled by a dense T-cell layer. Within the T-cell layer, compact B-cell aggregates were observed in most TB samples. These B-cell clusters were vascularized and could contain defined B-/T-cell and macrophage-rich areas. The ISS of 40–60 immune transcripts revealed the enriched expression of transcripts involved in homing or migration to lymph nodes, which formed networks at single-cell distances in lymphoid areas of the TB lesions. Instead, myeloid-annotated regions were enriched in CD68, CD14, ITGAM, ITGAX, and CD4 mRNA. CXCL8 and IL1B mRNA were observed in granulocytic areas in which M. tuberculosis was also detected. In line with ISS data indicating tertiary lymphoid structures, immune labeling of TB sections expressed markers of high endothelial venules, follicular dendritic cells, follicular helper T cells, and lymph-node homing receptors on T cells. Neither ISS nor immunolabeling showed evidence of tertiary lymphoid aggregates in sarcoidosis samples. Together, our finding suggests that despite their heterogeneity, the formation of tertiary immune structures is a common feature in granulomas from TB patients.</p