Colocalization of CD4⁺ and CD8⁺ lymphocytes with F4/80⁺ macrophages increases as infection with <i>Salmonella</i> persists.

<p>Mice were infected i.v. with 1x10⁵ CFUs of <i>Salmonella</i> and at day 0 (uninfected control), 6, 9, and 21, spleen tissue sections were stained with antibodies specific for CD4 and CD8 lymphocyte markers, as well as the F4/80 macrophage marker. Stained sections were analyzed with Volocity software and data are expressed as the percentage of pixels positive for the CD4 or CD8 marker colocalizing with pixels positive for the F4/80 cell marker (A). (B) Percentage of F4/80⁺ pixels colocalizing with CD4⁺ and CD8⁺ pixels. (C) A representative image of in situ colocalization of CD4⁺ lymphocytes (top panel, green) with F4/80⁺ macrophages (top panel, red) and CD8⁺ lymphocytes (bottom panel, green) with F4/80⁺ macrophages (bottom panel, red). Five images from individual mice were analyzed (n = 3 mice per time point). Data are expressed as the mean ± standard deviation. Group means that do not share a subscript are significantly different (p< 0.05).</p

Increased proportions of F4/80⁺ splenic RP macrophages during <i>Salmonella</i> infection.

<p>C57BL/6 mice were infected with 5x10⁵ CFU (high dose) or 1x10⁵ CFUs (low dose) of <i>Salmonella</i> and were euthanized 3, 6, 9, 14, and 21 days post-infection along with uninfected controls (day 0). (A) Images were analyzed using Volocity software and proportions of F4/80⁺ cells are expressed as the percentage of pixels (image area) per total image area from 5 images per mouse (n = 3 mice per time point). (B) Single cell suspensions from spleens of C57BL/6 mice infected with 1x10⁵ CFUs of <i>Salmonella</i> and uninfected controls (day 0) were stained with antibodies specific for the F4/80 cell marker and analyzed by flow cytometry. Data are expressed as the mean ± standard deviation (n = 3 mice per time point). Group means that do not share a superscript are significantly different from each other (p< 0.05). (C) In situ changes in the distribution of F4/80⁺ macrophages (red) at day 0 (control), 6, and 9 post-infection with 1x10⁵ CFUs of <i>Salmonella</i>. Actin staining with phalloidin-Alexa350 (green) highlights the tissue architecture of the spleen.</p

Infection with <i>Salmonella</i> causes marked increase in spleen size and alters the proportions of erythroid cells in the spleens of C57BL/6 mice.

<p>(A) Spleens of uninfected control mice (day 0) and mice infected with 1x10⁵ CFUs of <i>Salmonella</i> were excised and weighed at 0, 6, 9, 14, and 21 days post-infection. Spleen weights are shown as a percentage of body weight. Data are expressed as the mean ± standard deviation (n = 3 mice per time point). (B-D) Spleen tissue sections were stained with panels of 3 fluorescently tagged monoclonal antibodies and images were analyzed using Volocity software. The percentage of image area containing cells expressing erythroid cell markers was calculated from 5 images per mouse, per cell marker (n = 3 mice per time point). (B) Proportions of CD71⁺, Ter119⁺, and (C) CD71⁺ Ter119^-, CD71^- Ter119⁺, CD71⁺ Ter119⁺ erythroid cell populations are expressed as the mean ± standard deviation (n = 3 mice per time point). Group means that do not share a superscript are significantly different from each other (p< 0.05). (D) Representative images showing CD71⁺ (top row, red) and Ter119⁺ (bottom row, red) erythroid cells in spleen sections at day 0 (control), 3, 9, and 21 post-infection. Actin staining with phalloidin-Alexa350 (green) highlights the tissue architecture.</p

Colocalization of F4/80⁺ macrophages with RBCs and their immature precursors during infection with <i>Salmonella</i>.

<p>C57BL/6 mice were infected i.v. with 1x10⁵ CFUs of <i>Salmonella</i> and spleen tissue sections were stained with antibodies specific for CD71, Ter119, and F4/80 cell markers. Two (A) and three color (B) colocalization analysis was performed using Volocity software. Data are expressed as the percentage of pixels positive for CD71 or Ter119 colocalizing with pixels positive for F4/80 (A) or as the percentage of pixels positive for CD71 and/or Ter119 colocalizing with pixels positive for the F4/80 cell marker (B). Data are expressed as the mean ± standard deviation of 5 images analyzed per mouse (n = 3 mice per time point). Group means that do not share a superscript are significantly different from each other (p< 0.05). (C) A representative 3 color image showing colocalization of CD71⁺ (green, panel 1) and Ter119⁺ (purple, panel 2) cells with F4/80⁺ (red, panel 3) macrophages. Panel 4 shows overlap of panels 1–3.</p

Infection with <i>Salmonella</i> leads to the destruction of B cell follicles in the spleen and diminishes the proportions of B220⁺ lymphocytes.

<p>C57BL/6 mice were infected i.v. with 5x10⁵ CFUs (high dose) or 1x10⁵ CFUs (low dose) of <i>Salmonella</i> and at 0 (uninfected control), 3, 6, 9, 14, and 21 days post-infection, spleen sections were stained with monoclonal antibodies specific for the B220 cell marker. (A) Five images per mouse spleen (n = 3 mice per time point) were analyzed using Volocity software and the image area containing B220⁺ lymphocytes (red) was expressed as a percent of the total image area. (B) Spleen single-cell suspensions were stained with antibodies specific for the B220 cell marker and analyzed by flow cytometry. Data are expressed as the mean ± standard deviation (n = 3 mice per time point. The mean value for day 14 is averaged from 2 mice. Group means that do not share a superscript are significantly different from each other (p< 0.05). (C) Representative images of spleen tissue sections from uninfected mice (day 0) or mice infected with 5x10⁵ CFUs of <i>Salmonella</i> (B220⁺ B cells, red). Actin staining with phalloidin-Alexa350 (green) highlights the tissue architecture. (D) Representative flow cytometry contour plots showing proportions of B220⁺ splenocytes at day 0, 6, 9, and 14 post-infection.</p

Infection with <i>Salmonella</i> leads to decreased proportions of CD4⁺ and CD8⁺ T lymphocytes in the spleen.

<p>Spleens of C57BL/6 mice infected with 1x10⁵ CFUs of <i>Salmonella</i> were stained with antibodies specific for CD4 and CD8 cell markers. (A) Images were analyzed using Volocity software and the image area (pixels) containing CD4⁺ or CD8⁺ T lymphocytes were expressed as a percentage of the total area from 5 images per mouse spleen (n = 3 mice per time point). (B) Proportions of CD4⁺ and CD8⁺ splenocytes in cell suspensions of C57BL/6 mice infected with 1x10⁵ CFUs of <i>Salmonella</i> at day 0 (control), 6, 9, and 21 days post-infection analyzed by flow cytometry. Data are expressed as the mean ± standard deviation (n = 3 mice per time point). Group means that do not share a superscript are significantly different from each other (p< 0.05). (C) Representative images of spleens from control mice and mice infected with 1x10⁵ CFUs of <i>Salmonella</i> showing changes in the proportions of CD4⁺ (top row, red) or CD8⁺ cells (bottom row, red). Actin staining with phalloidin-Alexa350 (green) highlights the tissue architecture.</p

Infection with <i>Salmonella</i> causes anemia and alters proportions of erythroid cell populations in peripheral blood.

<p>C57BL/6 mice were infected i.v. with 1x10⁵ CFUs of <i>Salmonella</i> and at day 0 (uninfected control), 9, 14, and 21, blood samples were collected. (A) PCV (%) was calculated by dividing packed cell volume by total blood volume. (B) Blood samples were stained with fluorescent antibodies specific for erythroid cell markers CD71 and Ter119, then analyzed by flow cytometry. Proportions of CD71⁺ Ter119^-, CD71⁺ Ter119⁺, and CD71^- Ter119⁺ reticulocytes are shown as means ± standard deviation (n = 3 mice per time point). (C) Representative flow cytometry contour plots of blood samples taken at day 0, 9, 14, and 21 post-infection. Group means that do not share superscripts are significantly different from each other (p< 0.05).</p

Decreased proportions of MOMA⁺ MZ macrophages during infection with <i>Salmonella</i>.

<p>C57BL/6 mice were infected i.v. with (A) 5x10⁵ CFUs (high dose) or (B) 1x10⁵ CFUs (low dose) of <i>Salmonella</i> and spleen sections were stained with MOMA antibodies. Images were analyzed using Volocity software and data are expressed as the percentage of pixels positive for the MOMA cell marker per total image area. Data is expressed as the mean ± standard deviation. Means within a group that do not share superscripts are significantly different from each other (p< 0.05). (C) Representative images showing the changes in the distribution of MOMA⁺ macrophages (red) at day 0 (control), 6, and 9 post-infection in spleens of mice infected with 5x10⁵ CFUs of <i>Salmonella</i>. Actin staining with phalloidin-Alexa350 (green) highlights the tissue architecture of the spleen.</p

TEM images of spleen sections of <i>Salmonella</i>-infected mice.

<p>(A) A macrophage (Mϕ) with two phagocytosed lymphocytes (L). (B) A macrophage wrapping itself around two lymphocytes. (C) A macrophage with two phagocytosed lymphocytes. (D) A macrophage with a phagocytosed lymphocyte and RBC. (E) A lymphocyte within a macrophage’s cytoplasm. (F) A macrophage that has phagocytosed a lymphocyte.</p

Colocalization of B220⁺ lymphocytes with F4/80⁺ macrophages increases as infection with <i>Salmonella</i> persists.

<p>Mice were i.v. infected with 1x10⁵ CFUs of <i>Salmonella</i> and at day 0 (uninfected control), 6, 9, and 21, spleen tissue sections were stained with antibodies specific for the B220 lymphocyte marker and F4/80 macrophage marker. Stained sections were analyzed with Volocity software and data are expressed as the percentage of pixels positive for the B220 marker colocalizing with the pixels positive for the F4/80 cell marker (A). (B) Percentage of F4/80⁺ pixels colocalizing with B220⁺ pixels. (C) A representative image of in situ colocalization of B220⁺ lymphocytes (green) with F4/80⁺ macrophages (red). Five images from each individual mouse were analyzed (n = 3 mice per time point). Data are expressed as the mean ± standard deviation. Group means that do not share a subscript are significantly different (p<0.05).</p