IRF8-deficient microglia have a marked reduction in ramification and surface area.

<p>Representative confocal images of eGFP positive WT (A) and IRF8-deficient (B) microglia in the cortex, counterstained with DAPI (blue) were obtained from 100 µm vibratome sections on a Zeiss LSM 510 Meta confocal microscope. The 3D analysis (surface rendering) was performed using Bitplane Imaris software. Size bar = 10 µm. Quantification of single-cell surface area (C) and volume (D) for microglia in the cortex of WT (n = 5) or IRF8-deficient (n = 7) mice. Values are shown with the bar representing the mean. IRF8-deficient microglia showed a significant (p<0.0025; Mann-Whitney U test) reduction in cell surface area and although cellular volume of these cells was increased slightly this was not significant.</p

GFP⁺ cells in the CNS are exclusively CD11b⁺ microglia.

<p>Flow cytometry was performed on cells isolated from the brain of healthy adult WT (A, B) and IRF8-deficient mice as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049851#s4" target="_blank">Materials and Methods</a>. For analysis, cells were scatter-gated to exclude dead cells and GFP⁺ cells were selected (A). Greater than 99% of GFP⁺ cells expressed CD11b in WT and IRF8-deficient mice (B), consistent with a microglial phenotype. Isotype matched antibodies were used to determine background staining (data not shown).</p

Comparative features of cultured microglia from WT versus IRF8-deficient mice and localization of IRF8 in the brain.

<p>Microglial cell cultures were prepared from the brain of neonatal mice as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049851#s4" target="_blank">Materials and Methods</a>. Western blot analysis was performed on lysates of WT and IRF8-deficient primary microglia following treatment with (100 U/ml) or without IFN-γ for 4 h. GAPDH was used as a loading control (A). Morphological appearance of WT (B) or IRF8-deficient (C) microglia in primary culture (original magnification panels B&C, 400X). Immunohistochemical detection of IRF8 (D & E, arrows) combined with histochemical staing for tomato lectin binding (D) or GFAP immunohistochemistry (E, arrowheads). performed on brain sections from healthy adult mice. IRF8 staining is mostly confined to the nucleus of the cells (original magnification panel D, 1000X).</p

The Role of CXCR3 in DSS-Induced Colitis

<div><p>Inflammatory bowel disease (IBD) is a group of disorders that are characterized by chronic, uncontrolled inflammation in the intestinal mucosa. Although the aetiopathogenesis is poorly understood, it is widely believed that IBD stems from a dysregulated immune response towards otherwise harmless commensal bacteria. Chemokines induce and enhance inflammation through their involvement in cellular trafficking. Reducing or limiting the influx of these proinflammatory cells has previously been demonstrated to attenuate inflammation. CXCR3, a chemokine receptor in the CXC family that binds to CXCL9, CXCL10 and CXCL11, is strongly overexpressed in the intestinal mucosa of IBD patients. We hypothesised that CXCR3 KO mice would have impaired cellular trafficking, thereby reducing the inflammatory insult by proinflammatory cell and attenuating the course of colitis. To investigate the role of CXCR3 in the progression of colitis, the development of dextran sulfate sodium (DSS)-induced colitis was investigated in CXCR3^−/− mice over 9 days. This study demonstrated attenuated DSS-induced colitis in CXCR3^−/− mice at both the macroscopic and microscopic level. Reduced colitis correlated with lower recruitment of neutrophils (<i>p = </i>0.0018), as well as decreased production of IL-6 (<i>p</i><0.0001), TNF (<i>p</i> = 0.0038), and IFN-γ (<i>p</i> = 0.0478). Overall, our results suggest that CXCR3 plays an important role in recruiting proinflammatory cells to the colon during colitis and that CXCR3 may be a therapeutic target to reduce the influx of proinflammatory cells in the inflamed colon.</p></div

Surface levels of various molecules are altered on IRF8-deficient microglia.

<p>Flow cytometry (A-J) was performed on microglial cells isolated from the brain of adult WT (black dashed line) and IRF8-deficient (black line) mice as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049851#s4" target="_blank">Materials and Methods</a>. For analysis, cells were scatter-gated to exclude dead cells and were selected for GFP, CD11b expression. An isotype matched antibody was used as a negative control (grey dashed line). Quantification of forward and sideward scatter; (K) or mean fluorescent intensity of histograms (L). The histograms represent means +/− SD from three separate experiments. For significance: ***P<0.001, **P<0.01, *P<0.05; by two-tailed t-test.</p

The levels of some key myeloid markers are altered in the brain of IRF8-deficient mice.

<p>Immunostaining was performed on brain sections from healthy, adult WT (A–D) or IRF8-deficient (E–H) mice as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049851#s4" target="_blank">Materials and Methods</a>. Panels A, B, C, E, F, G show cortex while panels D, H show cerebellum (original magnification all panels 1000X). For immunofluorescence (G, H) DAPI was used to stain nuclei. Whole brain lysates were prepared from healthy, adult mice and 20 µg of protein analysed by western blotting (I).</p

Transcriptional transactivation of HSV-1 IE gene expression by HSV-2 helper virus.

<p>(A) Replication of HSV-2 in TGEs. Groups of three TGEs were either infected in the AC with 5×10⁶ pfu of HSV-2 333 (grey bars) or in the GC with 10⁴ pfu of HSV-2 333 (black bars), and harvested at the time points indicated. DNA extracts were pooled, genome levels were determined by qPCR, and the increase in genome levels relative to 1 hpi was calculated. (B, C) Transactivation of HSV-1 by HSV-2 infection of the GC. TGEs were infected in the AC with 5×10⁶ pfu of HSV-1 CMV-IEproEGFP, and infected either simultaneously or at 7 dpi in the GC with 5×10⁶ pfu of HSV-2 333. Cultures were harvested at 24 hpi after the addition of HSV-2 to the GC, and HSV-1 genome (B) and relative EGFP transcript levels (C) were determined. Cultures not infected with helper virus served as controls. The statistical significances of differences in the transcript levels are indicated (simultaneous infection, <i>P</i><0.0001; addition of PrV at 7 dpi, <i>P</i><0.0001; Mann-Whitney test). (D) Transcriptional transactivation of HSV-1 by co-infection of the GC with HSV-2 in the presence of CHX. TGEs were infected in the AC with 5×10⁶ pfu of HSV-1 17 IE4proEGFP in the presence of CHX and either co-infected in the GC with 5×10⁶ pfu of HSV-2 333 or mock-treated. Cultures were harvested at 6 hpi and EGFP transcript levels normalized to β-actin transcripts were determined. The statistical significances of differences in the mean relative transcript levels are indicated (<i>P</i>>0.0001, unpaired t-test with Welch correction).</p

Transcriptional transactivation of HSV-1 IE gene expression by PrV helper virus.

<p>(A) Replication of PrV in TGEs. Groups of three TGEs were either infected in the AC with 5×10⁶ pfu of PrV-KaDgGgfp (grey bars) or in the GC with 10⁴ pfu of PrV-KaDgGgfp (black bars), and harvested at the time points indicated. DNA extracts were pooled, genome levels were determined by qPCR, and the increase in genome levels relative to 1 hpi was calculated. (B) Transcriptional transactivation of HSV-1 by PrV infection of the GC. TGEs were infected in the AC with 5×10⁶ pfu of HSV-1 CMV-IEproEGFP, and infected either simultaneously or at 7 dpi in the GC with 5×10⁶ pfu of PrV-KaDgGgfp. Replication of PrV was suppressed by the addition of 50 µg/ml ACV to the media. Cultures were harvested at 6 hpi after the addition of PrV to the GC, and relative ICP27 transcript levels were determined. Cultures not infected with helper virus served as controls. The statistical significances of differences in the transcript levels are indicated (simultaneous infection, <i>P</i><0.0001; addition of PrV at 7 dpi, <i>P</i><0.0001; unpaired <i>t</i> test with Welch correction). (C) Transcriptional transactivation of HSV-1 by co-infection of the GC with PrV in the presence of CHX. TGEs were infected in the AC with 5×10⁶ pfu of HSV-1 17 CMV-IEproEGFP in the presence of CHX and either co-infected in the GC with 5×10⁶ pfu of PrV-KaΔgGgfp or mock-treated. Cultures were harvested at 6 hpi and ICP27 transcript levels normalized to β-actin transcripts were determined. The statistical significances of differences in the mean relative transcript levels are indicated (<i>P</i> = 0.0186, unpaired <i>t</i> test with Welch correction). (D) Transcriptional transactivation of HSV-1 by co-infection of the AC with PrV. TGEs were infected in the AC with 2.5×10⁶ pfu of HSV-1 17 syn⁺ in the presence or absence of CHX and co-infected in the AC with 2.5×10⁶ pfu of PrV-KaΔgGgfp or mock-treated as indicated. Cultures were harvested at 6 hpi and ICP27 transcript levels normalized to β-actin transcripts were determined. The statistical significances of differences in the mean relative transcript levels are indicated (mock+CHX vs. PrV+CHX, <i>P</i>>0.999; mock+CHX vs. PrV−CHX, <i>P</i><0.0001; PrV+CHX vs. PrV−CHX, <i>P</i><0.0001; Mann-Whitney test), <i>ns</i> (not significant).</p

Transactivation of HSV-1 in axonally-infected TGEs by infection of the GC with HSV-1 helper virus.

<p>(A) Effect of helper virus on EGFP expression in TGEs infected in the AC with HSV-1 17 CMV-IEproEGFP. TGEs were co-infected with 1×10⁶ pfu of the gH-negative, spread-deficient mutant HSV-1 KOS gH87 in the GC and 5×10⁶ pfu of HSV-1 17 CMV-IEproEGFP in the AC. At 1 hpi, cultures were stained with DiI in the AC. The arrowhead indicates a typical DiI and EGFP double-positive neuron depicted at higher magnification in the right-hand images. (B) Effect of helper virus on EGFP expression in axonally-infected neurons. The numbers of positive neurons/culture infected in the AC with 5×10⁶ pfu of HSV-1 17 CMVpro-IE EGFP and co-infected in the GC with 5×10⁶ pfu of HSV-1 KOS gH87 (ΔgH) or mock-infected are given. Differences were highly significant (<i>P</i> = 0.0003, unpaired <i>t</i> test with Welch correction). (C) Effect of helper virus on genome replication of HSV-1 after infection of the AC. Groups of ten cultures were infected in the AC with 5×10⁶ pfu of HSV-1 17 CMV-IEproEGFP. The GC was co-infected with varying amounts of HSV-1 KOS gH87, as indicated. TGEs were harvested at 24 hpi with the helper virus, reporter virus genome levels were quantified by qPCR, and the increase of reporter virus genome levels relative to controls was calculated. The significances of helper-virus-induced increases in the median genome level of reporter virus genomes are indicated (5×10⁶ pfu helper virus, <i>P</i><0.0001; 1×10⁶ pfu helper virus, <i>P</i> = 0.0021; 2×10⁵ pfu helper virus, <i>P</i> = 0.0015; 4×10⁴ pfu helper virus, <i>P</i> = 0.4359; Mann-Whitney test). Data are mean and SD values. (D) Reporter gene expression 24 h after co-infection of the GC with HSV-1 helper virus. Cultures were infected with 5×10⁶ pfu of HSV-1 17 CMV-IEproEGFP, and HSV-1 17 gDproEGFP in the AC. Groups of ten cultures were co-infected with 5×10⁶ pfu of HSV-1 KOS gH87 in the GC; cultures not infected with helper virus served as controls. The significances of differences in the relative transcript levels are indicated (HSV-1 CMV-IEpro EGFP, <i>P</i><0.0001; HSV-1 gDproEGFP, <i>P</i><0.0001; unpaired <i>t</i> test with Welch correction). (E) Helper-virus-induced transcriptional transactivation of IE gene expression in the absence of <i>de novo</i> protein synthesis. Cultures were infected in the AC with 5×10⁶ pfu of HSV-1 17 IE4proEGFP in the presence of 50 µg/ml CHX. Groups of ten cultures were co-infected in the GC with 5×10⁶ pfu of HSV-1 KOS gH87; cultures without helper-virus co-infection served as controls. TGEs were harvested in the AC at 6 hpi and the relative transcript levels of EGFP were determined. The statistical significances of differences are indicated (<i>P</i> = 0.0003; unpaired <i>t</i> test with Welch correction). (F–H) Effect of HSV helper virus added to the GC of the AC at 7 dpi. (F, G) Cultures were infected in the AC with 5×10⁶ pfu of HSV-1 17 CMV-IEproEGFP. At 7 dpi, cultures were infected in the GC with 5×10⁶ pfu of HSV-1 KOS gH87, cultures without helper-virus infection served as controls. At 24 h after the addition of helper virus, TGEs were harvested and genome levels of the reporter virus (F) and EGFP transcript levels were determined. There were no significant differences in reporter virus genome and transcript levels (genomes, <i>P</i>>0.9999, Mann-Whitney test; transcript levels, <i>P</i> = 0.7609, unpaired <i>t</i> test with Welch correction). (H) Cultures were infected in the AC with 5×10⁶ pfu of HSV-1 17 IE4proEGFP. At 7 dpi, cultures in the AC were either infected in the GC with 5×10⁶ pfu of HSV-1 KOS gH87 or mock-infected in the presence of CHX, and EGFP transcript levels were determined 6 h after addition of the helper virus. There were no significant differences in transcript levels (<i>P</i> = 0.8269, unpaired <i>t</i> test with Welch correction).</p

Detection of primary infected cells in dispersed TGEs at 2 dpi.

a<p>TGEs were infected either in the axonal or ganglion compartment (AC, GC).</p>b<p>Cultures were infected with HSV-1 17syn⁺ in the presence of 50 µg/ml ACV, harvested at 2 dpi and dispersed.</p>c<p>Genome levels per dispersed TGE were quantified by qPCR (mean of three experiments).</p>d,e<p>HSV-1-positive cells and neurons per dispersed TGE were detected and quantified by immunofluorescence with a HSV-1-specific rabbit antiserum (d), a monoclonal antibody against the 200 kD neurofilament marker (e) and DAPI (d,e) (mean of three experiments).</p