Frequency of splenic B, T, NK, and NKT cell subsets in untreated or MCMV-infected WT or TLR9^−/− male and female mice.

<p>WT and TLR9^−/− male and female mice were left uninfected or infected i.p. with 1,2×10⁵ PFU of MCMV. After, 36 h spleens were harvested, total splenocytes were isolated, stained for B220, CD19, CD3, CD4, CD8, or NK1.1 and analyzed by flow cytometry. The frequency of B cells (CD19⁺B220⁺), CD4 (CD3⁺CD4⁺) and CD8 (CD3⁺CD8⁺) T cells, and NK (B220⁻NK1.1⁺CD3⁻), and NKT (B220⁻NK1.1⁺CD3⁻) cells was determined after gating among live cells. All values denote percentages (mean ± SD) of three mice per group, and are representative of two or three independent experiments.</p

Increased proportion of splenic pDCs in MCMV-infected WT male mice.

<p>WT and TLR9^−/− male and female mice were left uninfected or infected i.p. with 1×10⁵ PFU of MCMV and spleens were harvested 36 h later. Erythrocyte-depleted splenocytes were analyzed by flow cytometry for the expression of B220, CD11c and Ly6C. Upon MCMV-infection the percentage of pDCs (B220⁺Ly6C^hiCD11c^lo) is increased in all four mouse groups, while the percentage of cDCs (B220⁻Ly6C⁻CD11c^hi) is slightly decreased. Interestingly, upon MCMV infection WT male mice showed statistically significant increased percentage of pDCs compared to WT females. Percentages on pDCs and cDCs are presented as mean ± SEM *p<0.05. The data are representatives of 3 mice per group and of 3 independent experiments.</p

Increased liver pathology in wild-type female compared to male mice infected with MCMV for 3 days.

<p>Wild-type male and female mice were left untreated (control) or infected with MCMV for 3 days and the following distinct measures of liver pathology were evaluated: necrotic foci, microscopic inflammatory foci and karyomegaly as revealed with H&E staining; and lipidosis as revealed by Oil red O staining. The grading system was as follows: for necrosis 1<5%, 2 = 5–10%, 3 = 10–20%, 4 = 20–40% and 5>40% of hepatocellular lobule affected; for inflammation 1 = mild, 2 = moderate, 3 = marked numbers of inflammatory cells; and lipidosis 1 = few lipid vacuoles of small size, 2 = moderate numbers of lipid vacuoles of small to medium size and 3 = numerous clear vacuoles ranging from small to very large size in hepatocytes.</p

Differences in the proportion of MZ B cells in MCMV-infected WT male and female mice.

<p>WT and TLR9^−/− male and female mice were left uninfected or infected i.p. with 1×10⁵ PFU of MCMV and spleens were harvested 36 hours later. Erythrocyte-depleted splenocytes were analyzed by flow cytometry for the expression of B220, CD19, CD23, CD21, IgM and IgD. The MZ B cell population, B220⁺CD19⁺CD23^loCD21^hi on (A) or IgM^highIgD^lowCD21^high on (B), of MCMV-infected WT male and TLR9^−/− male and female mice was dramatically reduced compared to uninfected counterparts, while this reduction was minor in MCMV-infected female mice. (A) Plots show percentage of MZ B cells (CD23^loCD21^hi) on B220⁺ gated lymphocytes. (B) Histograms show expression of CD21 on IgM^highIgD^low B cells. (C) Graph with the numeric data of all 3 mice per group that were used for the experiments in (A) and (B). *p<0.05. The data are representatives of 3 independent experiments.</p

Sequences of Primers used for ChIP and gene expression analysis

<p>Sequences of Primers used for ChIP and gene expression analysis</p

Increased number of neutrophils in the splenic white pulp of MCMV-infected WT male mice.

<p>WT or TLR9^−/− male and female mice were left untreated of infected with 1×10⁵ PFU of MCMV and 4 days later spleens were collected and processed for immunofluorescent analysis. (A) Murine spleen sections were incubated with antibodies specific to neutrophils (7/4, blue or red), B cells (B220, red), marginal metalophilic macrophages (MOMA-1, green) and reticular fibroblasts (ER-TR7, green). MCMV infection led to (A) a dramatic increase in the number of neutrophils that were present in the splenic red pulp and disappearance of the MZ metallophilic macrophages and (B) an increase in the number of neutrophils in the splenic white pulp areas compared to uninfected mice. These phenomena were more prominent in WT male than in WT female or TLR9^−/− male and female mice. (B) Number of neutrophils per white pulp area were counted on slides stained in A. *** p<0.001. Data are representative of 9 mice per group.</p

Analysis of Polycomb EZH2, M33 and BMI1 binding at the <i>INK4a/ARF</i> region.

<p>A) qPCR analysis of p16^INK4a and p19^ARF in young (P3), senescent (P10) and in <i>Bmi1</i> and <i>M33</i> mutant MEFs. B) B-galactosidase staining to detect senescent cells at passage 3 (P3) and passage 10 (P10). C) qPCR analysis of the mRNA levels of Polycomb EZH2 and Bmi1 in the indicated cells. D–F) Schematic diagram of the <i>INK4a/ARF</i> locus: amplified regions that were tested in ChIP experiments are indicated by red bars (sequences are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005622#pone-0005622-t001" target="_blank">Table 1</a>). Wild type P3 (young), P10–12 (senescent), M33−/− (P4) and Bmi−/− (P4) MEFs were subjected to ChIP assays using anti EZH2, BMI1 and M33 antibodies. DNA enrichment was calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005622#s2" target="_blank">Materials and Methods</a>. Bars represent the mean+/−s.d. of quantifications from two to four separate immunoprecipitations analyzed in triplicate.</p

Increased liver inflammation and steatosis in MCMV-infected female mice.

<p>(A) Livers were harvested and tissue sections were prepared from uninfected (A, C, E and G) or day 3 MCMV-infected male (B and F) and female (D and H) mice as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045171#s4" target="_blank">Materials and Methods</a>. Hematoxylin and eosin stained liver sections from uninfected (A and C) or MCMV infected (B and D) mice revealed inflammatory and necrotic foci in MCMV-infected mice. Female infected mice had increased inflammation and necrosis (more numerous and bigger foci) compared to male mice. Arrows identify inflammatory and necrotic foci, arrowhead shows an hepatocyte with an enlarged nucleous (karyomegaly) which contains large eosinophilic intranuclear inclusion and pheripheralized chromatin, asterisks indicate intracytoplasmic clear vacuoles. Accumulation of lipids (red stained lipid droplets) was detected by Oil red O staining (E–H) and was significantly more obvious in MCMV-infected female (H) versus male (F) liver sections, while in uninfected (E and G) mice was absent. Magnifications: A–D ×40 and E–H ×20. Data are representative of 2 independent experiments and with 3 mice per group.</p

Similar viral load and cytokine production in male and female mice.

<p>WT and TLR9^−/− male and female mice (n = 8–9) were infected i.p. with 1×10⁵ PFU of MCMV. (A) Four days after infection the viral load in spleen and liver was determined by Q-PCR with specific primers for IE-1 and GAPDH. For each genotype there is similar viral load between male and female mice, and TLR9^−/− mice showed increased viral load compared to WT counterparts. (B) Sera were collected 36 h after infection and the production of IL-6, TNFα, IL-12p70 and IFNγ were measured by CBA flex, and IFNα and IL-12p40 by ELISA. For each genotype there is similar cytokine levels between male and female mice, but statistically decreased cytokine levels in TLR9^−/− sera compared to WT mice. Data are representative of three independent experiments. *p<0.05, **p<0.01 and ***p<0.001.</p

Increased survival and splenic TLR9 expression of male mice upon MCMV infection.

<p>WT, TLR7^−/− and TLR9^−/− male (black circles) and female (white triangles) mice were infected i.p with (A) 1×10⁵ PFU or (B) 1.2×10⁵ PFU of MCMV and monitored twice daily for mortality. WT and TLR7^−/− male mice showed statistically increased survival compared to female counterparts, while no such sex difference was observed with TLR9^−/− mice. (C) WT male and female mice were infected i.p. with 1×10⁵ PFU of MCMV or left untreated. Spleens were harvested 36 h or 4 days later, total RNA was extracted and the expression of TLR7, TLR9 and β-actin mRNA was determined by Q-PCR. Splenic expression of TLR7 and TLR9 is increased 36 h upon MCMV infection, and TLR9 is expressed statistically significant higher in male than female mice. Data in (A) are representative of three independent experiments; in (B) and (C) two independent experiments have been pooled together. In (C) each point represents an individual mouse. *p<0.05; **p<0.0; ns, not significant; and n, number of mice per group.</p