CFH is constitutively expressed in the retina and up-regulated in RPE cells by IL-27 through STAT1-dependent mechanism.

<p>(A) Immunohistochemical localization of CFH expression in the mouse retina. Retina pigmented epithelium (RPE); Photoreceptors (pho); outer nuclear layer (ONL); outer plexiform layer (OPL); inner plexiform layer (IPL). (B) Whole cell extracts prepared from the retina or liver of C57BL/6 mice were analyzed for expression of CFH by Western blot assay. (C) ARPE-19 cells were stimulated for 24 hours in medium containing human IL-27 (10 ng/ml). ARPE-19 cells were cultured overnight in complete medium, washed and starved in serum free medium (SFM) for 2 hours and then stimulated in SFM containing human recombinant IL-27 (10 ng/ml) for 24 hours (C, D, E) or 30 minutes (F). (C) RNA was isolated and analyzed by qRT-PCR. (D) The ARPE-19 cells were stimulated with several concentrations of human IL-27 and then subjected to qRT-PCR analysis. (E, F) Whole cell extracts were analyzed by Western blot assay.</p

IL-27 induces expression of CFH through STAT1-mediated up-regulation of IRF-1 and IRF-8.

<p>(A–C) ARPE-19 cells were starved for 2 hours in serum free medium and cultures were then stimulated with IL-27 (10 ng/ml) for 30, 90 or 720 min. (B–D) In other cultures, cells were treated with CHX (35 ug/ml) for 30 min prior treatment with IL-27 for 30 min (B) or 24 h (C, D). Whole cell extracts were analyzed by Western blot assay and the antibodies used are indicated. (D) CFH mRNA transcripts were detected and quantified by qRT-PCR.</p

Expression of IRF-1, IRF-8 and IL-27 (IL27p28/EBi3) by retinal cells was up-regulated during experimental autoimmune uveitis (EAU).

<p>(A) Fundus images of the retina of un-immunized mouse and mouse with EAU. (B, C) Retinas were isolated from control or EAU mice and analyzed for IRF-1, IRF-8, CFH, IL27p28 or EBi3 expression by qRT-PCR.</p

Detection and localization of IRF-1 and IRF-8 proteins in the retina.

<p>Retinas were isolated from control or EAU mice and whole cell extracts were analyzed for IRF-1 (A) or IRF-8 expression by Western blot analysis. (C) Immunohistochemical localization of IRF-8 expression was detected in the normal mouse retina or retina of mice with EAU using an IRF-8-specific antibody as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045801#s2" target="_blank">Methods</a> section. Retina pigmented epithelium (RPE); Photoreceptors (pho); outer nuclear layer (ONL); outer plexiform layer (OPL); inner plexiform layer (IPL).</p

IL-27 induces CFH expression in the retina through STAT1-dependent mechanism.

<p>(A) Characterization of retina from mice with specific deletion of STAT3 in the retina (Ret-STAT3^−/−). (B) Freshly isolated retinas from wild type (WT), STAT1 knockout (STAT1^−/−), (Ret-STAT3^−/−), or from mice with targeted over-expression of SOCS1 in the retina (SOCS1Tg) were analyzed by RT-PCR. (C, D) Freshly isolated retinal cells from WT or STAT1^−/− mice were cultured overnight, washed, starved for 2 hours in serum-free medium and then cultured for an additional 22 hours in medium containing IL-27 (10 ng/ml). We analyzed RNA and total cell extracts from the cells by RT-PCR (C) or Western blotting (D).</p

Tm-TNF contributes to protective bactericidal granuloma formation during <i>M. tuberculosis</i> reactivation but is insufficient to sustain structural integrity and bactericidal efficacy.

<p>WT mice (A,D,G,J), TNF^−/− mice (B, E, H) and Tm-TNF mice (C,F,I,K) were infected by aerosol inhalation with 100–200 CFUs/mouse <i>M. tuberculosis</i> H37Rv for 3 weeks preceding chemotherapy with 25 mg/Kg INH-RIF for 6 weeks in drinking water. Lungs were removed at the indicated timepoints and tissue sections stained with haematoxylin and eosin to determine the granulomatous response.</p

Extra-granulomatous pulmonary expression of INOS is associated with susceptibility in Tm-TNF reactivating mice.

<p>WT mice (A,D), TNF^−/− mice (B) and Tm-TNF mice (C,E) were infected by aerosol inhalation with 100–200 CFUs/mouse <i>M. tuberculosis</i> H37Rv for 3 weeks preceding chemotherapy with 25 mg/Kg INH-RIF for 6 weeks in drinking water. Lungs were removed at 133 and 322 days post infection and tissue sections were stained with polyclonal rabbit anti-mouse antibody (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025121#s2" target="_blank"><i>Materials and methods</i></a>). Brown stain represents iNOS expression by activated macrophages. Micrographs represent 4 animals/group and are shown at ×32 magnification.</p

Graphic presentation of drug-based <i>M. tuberculosis</i> reactivation mouse model.

<p>Line A: Preimmune phase, short bacterial replication period post-infection with low dose <i>M. tuberculosis</i>; Line B: Steady state phase: Control of <i>M. tuberculosis</i> growth through host immunity and establishment of chronic infection. Line D: Drug treatment phase: Reduction of bacilli replication through INH-RIF chemotherapy. Line E: Reactivation phase: Reactivation of infection upon cessation of antibiotics.</p

Macrophage (CD11b⁺ cells) and lymphocyte (CD3⁺ cells) recruitment in WT and Tm-TNF mice.

<p>WT mice (A,C) and Tm-TNF mice (B,D) were infected by aerosol inhalation with 100–200 CFUs/mouse <i>M. tuberculosis</i> H37Rv for 3 weeks preceding chemotherapy with 25 mg/Kg INH-RIF for 6 weeks in drinking water. Lungs were removed 322 days post infection and tissue sections were stained with either anti-CD11b anti-mouse (A,B) antibody or anti-CD3 anti-mouse antibody (C,D) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025121#s2" target="_blank">Materials and methods</a>). Micrographs represent 4 animals/group and are shown at ×32 magnification.</p

Induction of excessive inflammation in the absence of solTNF during reactivation of <i>M. tuberculosis</i>.

<p>WT (black circles), TNF^−/− (white diamonds) and Tm-TNF (white squares) mice were exposed by aerosol inhalation infection to 100–200 CFUs/mouse of <i>M. tuberculosis</i> H37Rv for 3 weeks preceding chemotherapy with 25 mg/Kg INH-RIF for 6 weeks in drinking water. (A) Lung weights were measured at specific time points and BAL derived cell numbers were determined 77 days (B) and 378 days (C) post-infection. The red “T” in the figure corresponds to the drug treatment phase. Data are representative of 1 of 2 experiments performed and are expressed as mean ± SD of 5 mice/group. Significant differences (*<i>p</i><0.05; **<i>p</i><0.01; ***<i>p</i><0.001) were determined by Student's <i>t</i> test for comparisons between two groups and ANOVA for comparisons between three groups.</p