Effects of acute Theiler’s murine encephalomyelitis virus (TMEV)-infection (<i>experiment I</i>) upon cytokine expression and phenotypical changes in spleens of interleukin-10 receptor (IL-10R) blocked SJL mice.

<p>(A-I) Significantly elevated mRNA levels of IL-1α, IL-2, IL-4, IL-5, IL-6, TNF, IFN-γ, TGF-β and IL-10 in spleens of infected mice (group “IL-10R↓_early/TMEV”) compared to non-infected animals following IL-10R antibody (Ab) treatment (group “IL10R↓_early/mock”). (J) Flow cytometry revealed a relative increase of CD19⁺ B cells and a simultaneous decrease of CD4⁺ T cells (K) in the spleen of infected mice following IL-10R blockade at 14 dpi. (L) Additionally, the relative numbers of CD4⁺ Foxp3⁺ Treg in the spleen were increased following TMEV-infection and IL-10R blockade. Moreover, gMFI of CD69 (M) and CD44 (N) gated on CD4⁺ cells and gMFI of CD69 (O) and CD44 (P) gated on CD8⁺ cells were increased at 14 dpi in infected mice following IL-10R Ab treatment. For gating strategy see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161883#pone.0161883.s001" target="_blank">S1 Fig</a>. IL-10R Ab treated animals without TMEV-infection (group “IL10R↓_early/mock”). IL-10R Ab treated mice with additional TMEV-infection (group “IL-10R↓_early/TMEV”). Box and whisker plots display median, minimum and maximum values as well as upper and lower quartiles, 5 animals used in both groups and at all investigated time points, Wilcoxon rank-sum tests, *  =  p < 0.05.</p

Motor coordination in Theiler’s murine encephalomyelitis virus (TMEV)-infected SJL mice.

<p>(A) <i>Experiment I</i>: RotaRod^® performance test after early application of the IL-10 receptor blocking antibody (IL-10R Ab) at 0, 7, 14 and 21 dpi (arrows) revealed motor coordination deficits in TMEV-infected animals compared to mock-infected mice at 42 dpi. However, no differences were determined between IL-10R Ab- and isotype-treated animals. white box = TMEV-infected mice without IL-10R Ab treatment (group “isotype_early/TMEV”), grey box = TMEV-infected mice with IL-10R treatment (group “IL10R↓_early/TMEV”), black box = mock-infected mice with IL-10R treatment (group “IL10R↓_early/mock”). (B) <i>Experiment II</i>: application of IL-10R Ab at 35 and 42 dpi (arrows) caused also deterioration of motor coordination starting at 42 dpi. However, no differences were observed between IL-10R Ab- and isotype-treated animals (groups “IL-10R↓_late/TMEV” and “isotype_late/TMEV”). white box = TMEV-infected mice without IL-10R Ab treatment (group “isotype_late/TMEV”), grey box = TMEV-infected mice with IL-10R treatment (group “IL10R↓_late/TMEV”), black box = mock-infected mice with IL-10R treatment (group “IL10R↓_late/mock”). Box and whisker plots display median, minimum and maximum values as well as upper and lower quartiles, 5 animals used at all investigated time points, Wilcoxon rank-sum tests, *  =  p < 0.05.</p

Theiler’s murine encephalomyelitis virus (TMEV)-infection exacerbates enteric disease following interleukin-10 receptor (IL-10R) blockade.

<p>(A) Note changes of colonic mucosa with severe lymphohistiocytic to neutrophilic inflammation (arrow heads), crypt abscess formation (#), and loss of crypt epithelium (arrow). H&E staining, bar = 60 μm (B) Significantly increased severity of colitis at 14 days post infection (dpi) in TMEV-infected mice with IL10R antibody (Ab) treatment (group “IL10R↓_early/TMEV”) compared to Ab treated animals without infection (group “IL10R↓_early/mock”). Animals receiving isotype control instead of IL-10R Ab did not show any intestinal inflammation. IL-10R Ab treated SJL mice with TMEV-infection (group “IL10R↓_early/TMEV”), TMEV-infected mice without IL-10R Ab treatment (group “isotype_early/TMEV”), IL-10R Ab treated SJL mice without TMEV-infection (group “IL10R↓_early/mock”). Box and whisker plots display median, minimum and maximum values as well as upper and lower quartiles, 5 animals used in all groups, Wilcoxon rank-sum tests, *  =  p < 0.05.</p

Clinical effects of Theiler’s murine encephalomyelitis virus (TMEV)-infection in SJL mice following IL-10R blockade.

<p>(A) <i>Experiment I</i>: TMEV-infection causes worsening of systemic clinical signs (increased clinical scores) at 22 dpi in SJL mice with early anti-IL-10 receptor antibody (IL-10R Ab) treatment compared to non-infected mice following anti-IL-10R Ab treatment (<b>red asterisks</b>) and TMEV-infected mice without anti-IL-10R Ab treatment (<b>black asterisks</b>), suggestive of a triggering effect of virus infection upon IL-10R deficiency-mediated systemic signs. (B) <i>Experiment II</i>: Ab treatment during the late infection phase leads to similar clinical scores between TMEV-infected mice with and without IL-10R blockade. 5 animals used in all three groups and at all investigated time points, Wilcoxon rank-sum tests, arrows = administration of IL-10R Ab or isotype control, respectively, red asterisks = significant differences (p < 0.05) between TMEV-infected mice with and without IL-10R blockade, black asterisks = significant differences (p < 0.05) between IL-10R blocked mice with and without TMEV-infection, green asterisks significant differences (p < 0.05) between TMEV-infected mice and IL-10R blocked mice.</p

Summary of antibodies used for immunohistochemistry.

<p>Summary of antibodies used for immunohistochemistry.</p

Effect of IL-10 receptor (IL-10R) blockade upon leukomyelitis in Theiler’s murine encephalomyelitis virus (TMEV)-infected SJL mice.

<p>(A) TMEV-infection and concurrent intraperitoneal application of IL-10R antibody lead to an increase of CD3⁺ T cells in the spinal cord at 49 days post infection (dpi) compared to (B) TMEV-infected control animals. A,B: immunohistochemistry, bar = 20 μm. (C) Statistical analyses revealed a significant increase of CD3⁺ T cells in TMEV-infected mice with IL-10R blockade compared to isotype-treated animals at 49 dpi. Almost no CD3⁺ T cells were present in spinal cord cross sections of mock-infected animals. (D) Simultaneously, IL-6 mRNA expression significantly decreased in TMEV-infected animals following IL-10R blockade. white box = TMEV-infected mice without IL-10R Ab treatment (group “isotype_late/TMEV”), grey box = TMEV-infected mice with IL-10R treatment (group “IL10R↓_late/TMEV”), black box = mock-infected mice with IL-10R treatment (group “IL10R↓_late/mock”). Box and whisker plots display median, minimum and maximum values as well as upper and lower quartiles, 5 animals used in all groups, Wilcoxon rank-sum tests, *  =  p < 0.05.</p

Enteric disease following interleukin-10 receptor (IL-10R) blockade in non-infected SJL mice.

<p>(A) Unformed feces and mucosal edema in an animal at 14 days after onset of IL-10R antibody (Ab) treatment. (B) Colon of a control animal (group “isotype”) at day 21 with normal mucosal architecture. (C) Colon of an animal receiving IL-10R Ab (group “IL-10R↓”) at the same time point. Note infiltration of neutrophilic granulocytes (arrows) and loss of goblet cells (#). B, C: H&E staining, bar = 20 μm. (D) IL-10R blockade causes progressive colitis compared to control animals. grey box with vertical lines = control animals (group “isotype”), white box with vertical lines = IL-10R blocked animals (group “IL-10R↓”). Box and whisker plot display median, minimum and maximum values as well as upper and lower quartiles, 5 animals used at all investigated time points, Wilcoxon rank-sum tests, *  =  p < 0.05.</p

Effects of chronic Theiler’s murine encephalomyelitis virus (TMEV)-infection (<i>experiment II</i>) on cytokine expression and phenotypical changes in spleens of interleukin-10 receptor (IL-10R) neutralized SJL mice.

<p>(A-H) Significantly elevated mRNA levels of IL-1α, IL-2, IL-4, IL-6, TNF, IFN-γ, TGF-β and IL-10 in spleens of infected mice (group “IL-10R↓_late/TMEV”) compared to non-infected animals following IL-10R antibody (Ab) treatment (group “IL-10R↓_late/mock”). (I) Flow cytometry revealed a relative increase of CD8⁺ cells in the spleens of TMEV-infected mice with IL-10R Ab treatment at 49 dpi. For gating strategy see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161883#pone.0161883.s002" target="_blank">S2 Fig</a>. IL-10R Ab treated mice without TMEV-infection (group “IL-10R↓_late/mock”). IL-10R Ab treated mice with TMEV-infection (group “IL-10R↓_late/TMEV”). Box and whisker plots display median, minimum and maximum values as well as upper and lower quartiles, 5 animals used in both groups, Wilcoxon rank-sum tests, *  =  p < 0.05.</p

Summary of primer pairs used for polymerase chain reaction.

<p>Summary of primer pairs used for polymerase chain reaction.</p

Leukocyte subsets in the colon of interleukin-10 receptor (IL-10R) neutralized SJL mice.

<p>(A) Application of IL-10R antibody (Ab) leads to increased numbers of CD3⁺ T cells in the colon at all investigated time points. (B). An increase of CD45R⁺ B cells can be observed at day 21 after the first Ab application. (C) Foxp3⁺ regulatory T cells and (D) Iba-1⁺ macrophages are elevated at 7, 14, and 21 dpi. grey box with vertical lines = control animals (group “isotype”), white box with vertical lines = IL-10R blocked animals (group “IL-10R↓”). Box and whisker plot display median, minimum and maximum values as well as upper and lower quartiles, 5 animals used at all investigated time points, Wilcoxon rank-sum tests, *  =  p < 0.05.</p