Effect of deletion of <i>Ifng</i>, <i>Stat1</i>, <i>Jak3</i>, <i>Irf1</i>, <i>Irgm1</i>, <b><i>Il12p40</i></b>, <i>Ifit1</i>, <i>Isg15</i> and <i>Nlrc4</i> on susceptibility to PbA induced cerebral malaria.

<p>Control and mutant mice were infected with 10⁶<i>P. berghei</i> parasites and survival was monitored. Cerebral malaria susceptible mice succumbed between d5 and d10 post-infection with neurological symptoms, while no mice that survived longer than 13 days developed signs of ECM and these were categorized as resistant. Infection specific B6 and BXH2 controls (n>5 per infection) are plotted alongside each mutant strain.</p

<i>Irf8</i>-Regulated Genomic Responses Drive Pathological Inflammation during Cerebral Malaria

<div><p>Interferon Regulatory Factor 8 (IRF8) is required for development, maturation and expression of anti-microbial defenses of myeloid cells. BXH2 mice harbor a severely hypomorphic allele at <i>Irf8</i> (<i>Irf8^R294C</i>) that causes susceptibility to infection with intracellular pathogens including <i>Mycobacterium tuberculosis</i>. We report that BXH2 are completely resistant to the development of cerebral malaria (ECM) following <i>Plasmodium berghei</i> ANKA infection. Comparative transcriptional profiling of brain RNA as well as chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq) was used to identify IRF8-regulated genes whose expression is associated with pathological acute neuroinflammation. Genes increased by infection were strongly enriched for IRF8 binding sites, suggesting that IRF8 acts as a transcriptional activator in inflammatory programs. These lists were enriched for myeloid-specific pathways, including interferon responses, antigen presentation and Th1 polarizing cytokines. We show that inactivation of several of these downstream target genes (including the <i>Irf8</i> transcription partner <i>Irf1</i>) confers protection against ECM. ECM-resistance in <i>Irf8</i> and <i>Irf1</i> mutants is associated with impaired myeloid and lymphoid cells function, including production of IL12p40 and IFNγ. We note strong overlap between genes bound and regulated by IRF8 during ECM and genes regulated in the lungs of <i>M. tuberculosis</i> infected mice. This IRF8-dependent network contains several genes recently identified as risk factors in acute and chronic human inflammatory conditions. We report a common core of IRF8-bound genes forming a critical inflammatory host-response network.</p></div

Characterization of the lymphoid compartment in PbA-infected B6, BXH2, [BXH2×B6]F1 and <i>Irf1^−/−</i> mice.

<p>Characterization of the lymphoid compartment was carried out as described in the legend of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003491#ppat-1003491-g006" target="_blank">Figure 6</a>. Splenocytes were stained with CD45, TCRβ, CD4 and CD8 antibodies and representative cellular profiles are shown for each strain (A), where numbers indicate mean ± SD (gated as percentages of CD45⁺ cells). Absolute numbers are indicated in dot plots for total spleen CD4⁺ cells (B) and CD8⁺ cells (C). (D) Serum IFNγ levels were significantly lower in ECM-resistant BXH2, [BXH2×B6]F1 and <i>Irf1^−/−</i> mice. (E) IFNγ production was assayed <i>in vitro</i> in culture supernatants from infected mice with or without stimulation with PMA/Ionomycin or with IL12p70. The p-values were calculated relative to B6 controls with Student's t-test (**p<0.01, ***p<0.001).</p

Genes up-regulated during ECM pathology in B6 mice are significantly enriched for IRF8 binding sites.

<p>(A) Quantitative PCR was used to validate ChIP results using known binding targets of IRF8. Targets were highly enriched in IRF8-immunoprecipitated DNA when compared to control IgG preparations. Representative data from one of five independent experiments is shown. (B) The list of genes regulated by infection in B6 mice (d7/d0 pairwise) was interrogated for IRF8 binding sites within 225 kb of their transcription start site. The graph represents the abundance of IRF8 binding sites in each 25 kb segment. (C) IRF8 and control IgG ChIP-seq sequence reads were mapped to the mouse genome and significant IRF8 binding sites were identified. Light blue (top) track indicates non-specific (IgG) sequencing profile and dark blue track (below) displays IRF8 binding sites. Genes were considered to have an IRF8 binding site if a peak was found within 20 kb of the transcription start site.</p

BXH2 mice do not develop cerebral malaria following <i>Plasmodium berghei</i> infection.

<p>(A) Survival plots of <i>Plasmodium berghei</i> (PbA) infected BXH2 mice (n = 18), heterozygous [BXH2×B6]F1 offspring (n = 15), and ECM-susceptible B6 (n = 19) parental controls. Shown are the combined results of four experiments. (B) Blood parasitemia levels during infection for mice in Panel A following infection with PbA. (C) <i>Irf8</i> genotype-specific survival curves for 24 [BXH2×B6]F2 mice along with parental controls. (D) Qualitative comparison of representative Evans blue dyed brains from uninfected and infected B6 and BXH2 mice indicating breakdown of the blood-brain barrier in infected B6 (d7 PbA), but not BXH2 (d7 PbA or d16 PbA) mice. (E) Perfused brains from ECM-susceptible B6 and ECM-resistant BXH2 and [BXH2×B6]F1 mice (n = 2 to 4) were collected six days following infection with PbA. Infiltrating leukocytes were enriched by Percoll gradient and stained with CD45, Ly6C and CD11b, or CD45, TCRβ, CD4 and CD8 antibodies. The presence of myeloid and lymphoid infiltrates is observed in the brain of B6 mice compared to BXH2 or F1. Gate R3 denotes infiltrating cells gated by side scatter (SSC-A) and forward scatter (FSC-A).</p

Characterization of the myeloid compartment in PbA-infected B6, BXH2, [BXH2×B6]F1 hybrids and <i>Irf1^−/−</i>.

<p>Spleens from B6, BXH2, [BXH2×B6]F1 and IRF1^−/− mice were harvested prior to or six days following PbA infection, and processed by flow cytometry. 2×10⁶ splenocytes were stained with CD45, CD11b, Ly6C, Ly6G, F4/80, CD11c and MHCII, and representative cellular profiles are shown for each strain (A). The numbers within contour plots refer to gates R1 (monocytes) or R2 (granulocytes) and are reported as mean ± SD (gated as percentages of CD45⁺ cells). Absolute numbers of day 6 PbA-infected mice are shown for (B) CD11b⁺Ly6G⁺ and (C) CD11b⁺F4/80⁺ populations, indicating an expansion of the myeloid lineage in the BXH2 strain, compared to B6, [BXH2×B6]F1 and IRF1^−/− animals. Reduced numbers of myeloid dendritic cells (D) along with lower serum IL12p40 levels (E) are noted in ECM-resistant BXH2 and [BXH2×B6]F1 compared to ECM-susceptible B6 mice. (F) Levels of secreted IL12p40 were determined in culture supernatants from splenocytes of infected mice. Dashed gray line represents IL12p40 detection limit. Differences were considered significant when p<0.05 and calculated compared to the B6 strain (Student's t-test: *p<0.05, **p<0.01, ***p<0.001).</p

Specific Dysregulation of IFNγ Production by Natural Killer Cells Confers Susceptibility to Viral Infection

<div><p>Natural Killer (NK) cells contribute to the control of viral infection by directly killing target cells and mediating cytokine release. In C57BL/6 mice, the Ly49H activating NK cell receptor plays a key role in early resistance to mouse cytomegalovirus (MCMV) infection through specific recognition of the MCMV-encoded MHC class I-like molecule m157 expressed on infected cells. Here we show that transgenic expression of Ly49H failed to provide protection against MCMV infection in the naturally susceptible A/J mouse strain. Characterization of Ly49H⁺ NK cells from <i>Ly49h</i>-A transgenic animals showed that they were able to mount a robust cytotoxic response and proliferate to high numbers during the course of infection. However, compared to NK cells from C57BL/6 mice, we observed an intrinsic defect in their ability to produce IFNγ when challenged by either m157-expressing target cells, exogenous cytokines or chemical stimulants. This effect was limited to NK cells as T cells from C57BL/6 and <i>Ly49h</i>-A mice produced comparable cytokine levels. Using a panel of recombinant congenic strains derived from A/J and C57BL/6 progenitors, we mapped the genetic basis of defective IFNγ production to a single 6.6 Mb genetic interval overlapping the <i>Ifng</i> gene on chromosome 10. Inspection of the genetic interval failed to reveal molecular differences between A/J and several mouse strains showing normal IFNγ production. The chromosome 10 locus is independent of MAPK signalling or decreased mRNA stability and linked to MCMV susceptibility. This study highlights the existence of a previously uncovered NK cell-specific <i>cis</i>-regulatory mechanism of <i>Ifnγ</i> transcript expression potentially relevant to NK cell function in health and disease.</p></div

Decreased IFNγ production by Ly49H⁺ NK cells in A/J mice.

<p>Splenocytes from B6 and A-<i>Ly49h</i> mice were harvested and stimulated with RMAs-m157, BAF-m157, IL12/IL18 or PMA and ionomycin (P/I) for 3–5 h. (A) Representative dot plots demonstrating IFNγ production following stimulation and gaited on CD3⁻DX5⁺ Ly49H⁺ NK cells. The numbers represent the percentage of Ly49H⁺ producing IFNγ. (B) Graphical representation of IFNγ production by CD3⁻DX5⁺ Ly49H⁺ NK cells following stimulation. (C) Representative dot plots showing IFNγ production following stimulation and gaited on CD3⁺ or CD3⁻ cells. Numbers represent the percentage of cells producing IFNγ. (D) Graphical representation of IFNγ production by CD3⁻ DX5⁺ (T cells) or CD3⁺ DX5⁻ (NK cells) after P/I stimulation. Data were analyzed using two-tailed Student's <i>t</i>-test and presented as mean ± SEM and <i>P</i> values of significant results between groups are indicated. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

NK cells from A/J mice can proliferate and produce Granzyme B and Perforin following MCMV inoculums.

<p>Splenocytes were harvested from naïve B6 and A-<i>Ly49h</i> mice (day 0) or mice infected with 2500 PFU MCMV (n = 3 mice/time point). Time points post-infection are indicated in the figure. (A) BrdU incorporation was analyzed on CD3⁻DX5⁺ Ly49H⁺ NK cells by flow cytometry. (B) Spleen viral titers were determined by PA. Intracellular (C) Granzyme (D) Perforin expression was analyzed by flow cytometry on CD3-DX5+ Ly49H+ NK cells. Representative plots from individual mice are shown. The percent of Ly49H+ NK cells positive for Gzmb, and Prf1 are summarized for one experiment. Data were analyzed using two-tailed Student's <i>t</i>-test and presented as mean ± SEM and significant <i>P</i> values are indicated. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001. These data are representative of 2–3 independent experiments.</p

Mapping of IFNγ production by NK cells reveals a single locus on chromosome 10.

<p>(A) Genome-wide linkage analysis was done using mice from the 33 RCS strains outlined in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004511#ppat-1004511-g004" target="_blank">Figure 4A</a>. IFNγ production by NK cells following P/I treatment was used as the mapping trait. The negative log genome-wide <i>p</i> values are shown. (B) Chr 10 negative log genome-wide <i>p</i> values of IFNγ production by NK cells upon P/I treatment. (C) Map of the 6.6 Mbp relevant interval in chr 10 harboring 45 genes in black rectangles (adapted from UCSC mouse genome browser, mm9).</p