MyD88 deficiency does not completely abrogate NK cell-mediated protection.

<p>(A-D) Ly49H⁺ NK cell activation in BALB/c-Ly49H⁺, BALB/c-Ly49H⁺ MyD88^-/- and BALB/c-Ly49H⁺ IFNAR^-/- mice at d0, 3 and 6 post infection. Proliferation was assessed by Ki67 expression (A). Antiviral effector functions were assessed by intracellular staining for IFN-γ (B) and Granzyme B (C-D). (E) Impact of NK cell depletion on viral replication in the spleen of BALB/c-Ly49H⁺, BALB/c-Ly49H⁺ MyD88^-/- and BALB/c-Ly49H⁺ IFNAR^-/- mice. Mice were depleted of NK cells and splenic viral titers were measured 5 or 6d post infection. (F-I) Ly49H^- NK cell activation in BALB/c-Ly49H⁺, BALB/c-Ly49H⁺ MyD88, BALB/c-Ly49H⁺ IFNAR^-/-, BALB/c, BALB/c MyD88^-/- and BALB/c IFNAR^-/- mice. Data (mean±SEM) are shown from 3 pooled independent experiments each with 3 mice per group.</p

Model of redundancies and complementarities between molecular sensors and cell types for mounting the IFN-I, IFN-γ and cytotoxic cellular immune responses which are necessary for control of MCMV infection.

<p>Based on our own data as well as on many other studies published previously, we propose a model whereby IFN-I, IFN-γ and cytotoxic responses are all necessary for immune defenses against MCMV infection (red lines and text), but access to these functions can be promoted by a number of partly redundant and/or complementary pathways (arrows converging towards a number). IFN-I, IFN-γ and cytotoxic cellular immune responses are critical for control of viral replication, prevention of excessive tissue damage and overall resistance of the host in terms of morbidity and mortality. Several cell types and pathways can lead to IFN-I production. Early after intra-peritoneal injection, MCMV infects stromal cells and other cell types, inducing an IFN-I production by those cells independently of MyD88 and TLRs. In addition, viral particles or material derived from infected cells can be engulfed by cDC and pDC to promote their production of high levels of IFN-I or other cytokines upon triggering of the TLR7/9-to-MyD88 signaling cascade. In our experimental settings, MyD88/TLR9 responses of DC and MyD88-independent responses of infected cells are redundant for the induction of strong IFN-I responses, even though only very low to undetectable levels of IFN-I are produced in the absence of MyD88 responses (❶). In parallel, NK cells and CD8 T cells are able to produce IFN-γ and to specifically recognize and kill MCMV infected cells, via the activation receptor Ly49H or via their TCR, respectively. Cell-mediated immune control of viral replication can thus be performed by both NK and CD8 T cells which can largely compensate one another for this function (❷). However, the antiviral functions of NK and CD8 T cells are not strictly redundant but partly complementary, since, for example, the absence of CD8 T cell responses might increase the risk of selection of viral mutants able to escape NK cell control. MyD88 responses can promote the activation of NK and CD8 T cells via TLR-dependent activation of myeloid cells and/or through cell-intrinsic effects. However, MyD88 responses are not necessary for this function in mice expressing NK cell activation receptors able to directly sense MCMV-infected cells. In these mice, in addition to IFN-I production, MyD88-independent responses from infected cells might provide the other beneficial inflammatory signals necessary for this function (❸). Functional redundancies might also exist between MyD88 responses and NK cell activity for prevention of excessive damage to lymphoid tissues (❹), in order to preserve their micro-anatomical niches supporting the proliferation and survival of CD8 T cells.</p

pDC or MyD88 deficiency impairs IFN-I production but not splenic IFN-I responses and virus control.

<p>Control (Rat IgG), pDC-depleted (αBST2) or untreated BALB/c mice and untreated BALB/c, BALB/c MyD88^-/-, BALB/c IFNAR^-/- and BALB/c TLR9^-/- mice were infected with 2.5 x 10³ pfu MCMV or left uninfected (NI). (A) IFN-α serum titers were measured at d1.5 post-infection by ELISA. Results (mean±SEM) are shown from one experiment representative of 3 independent ones. (B) RT-PCR analysis of the expression of selected genes in the spleen at d1.5 or 3 post-infection. Data are normalized to mean expression of d1.5 BALB/c mice (100% reference level). Results (mean±SEM) are shown from 2 pooled independent experiments, each with 2 to 3 mice per group. (C) Serum IFN-α levels were measured at d1.5 post-infection by ELISA in BALB/c-Ly49H⁺, BALB/c-Ly49H⁺ MyD88^-/-, BALB/c and BALB/c MyD88^-/- mice. Results (mean±SEM) are shown from one experiment representative of 3 independent ones, each with 3 mice per group. BLD: below limit of detection. (D-E) Microarray analyses were performed on total mRNA extracted from spleen at d0, 1.5, 2, 3 and 6 post-infection. Heatmaps show the relative expression value for IFN-I/III genes (D) and for 25 ISG (E). The expression pattern of ISG is examined globally in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004897#ppat.1004897.s002" target="_blank">S2E</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004897#ppat.1004897.s002" target="_blank">S2G</a> Fig. Results shown are from 2 pooled independent experiments, each with 1 to 3 mice per group.</p

Impact of MyD88 or Ly49H deficiencies on antiviral CD8 T cell responses.

<p>(A-C) Analysis of antiviral CD8 T cell effector functions. (A) <i>In vivo</i> cytotoxicity, <i>ex vivo</i> IFN-γ production and Granzyme B expression by splenic CD8 T cells from BALB/c-Ly49H⁺, BALB/c-Ly49H⁺ MyD88^-/-, BALB/c and BALB/c MyD88^-/- mice at d0 and 6 post infection. Data (mean ± SEM) are shown from 3 pooled independent experiments each with 2 or 3 mice per group. (B-C) Impact of CD8 T cell depletion on disease in BALB/c and BALB/c MyD88^-/- mice. Mice were depleted of CD8 T cells and infected with 2.5x10³ pfu MCMV (B) or with 8x10³ pfu MCMV (C). Splenic viral titers were measured at d6 post infection (B) or mortality was monitored daily (C). For B, data (mean±SEM) are represented from 2 pooled independent experiments each with 2 to 4 mice per group. For C, data show the percent survival from 2 pooled independent experiments each with 3 to 5 mice per group, n represents the total number of mice per group. (D) Enumeration of total (left panel) and anti-IE-1 (right panel) CD8 T cells in the spleens of d5 MCMV-infected mice. Results (mean±SEM) are shown from 4 pooled independent experiments each with 3 mice per group. (E) Immunohistological analysis of tissue damage to the spleen in d4 MCMV infected mice. Mice were infected with 10⁴ pfu MCMV. Spleen were harvested at d4 and stained to evaluate the integrity of the T cell zone as assessed by examining its marginal zone boundary (CD169) and its T cell (CD3) and B cell (B220) zones. Results are shown for one representative mouse per experimental group from 3 independent experiments each with 3 mice per group.</p

IFN-I responses are essential but MyD88 and Ly49H partly redundant to protect against MCMV infection.

<p>(A) Survival of mice at d21 post-infection (Y-axis) as a function of the doses of MCMV inoculum (X-axis). Indicated mice were infected with between 1.6x10³ and 7x10⁴ PFU of MCMV, with overlaps for several doses between strains of mice, and their survival was followed for 21d. Data is shown as percentage of survival. In total, 8 different doses were tested in 9 different experiments in order to determine the susceptibility of each mouse strain, with 2 to 4 mouse strains simultaneously studied in each experiment. Data was derived from the following numbers of mice and tested doses of MCMV inoculum: BALB/c-Ly49H⁺: 24 mice for 4 doses; BALB/c-Ly49H⁺ MyD88^-/-: 37 mice for 6 doses; BALB/c-Ly49H⁺ IFNAR^-/-: 8 mice for 2 doses; BALB/c: 43 mice for 5 doses; BALB/c MyD88^-/-: 15 mice for 4 doses; BALB/c IFNAR^-/-: 6 mice for 2 doses. The median lethal dose that causes 50% of lethality in each mouse strain (LD₅₀) was calculated from the graph as the dose on the X-axis that corresponds to 50% mortality on the Y-axis as represented by the horizontal dotted line labeled “LD₅₀ calculation”. LD₅₀ BALB/c-Ly49H⁺: ≥10⁵ pfu/mouse; LD₅₀ BALB/c-Ly49H⁺ MyD88^-/-: 3.3x10⁴ pfu; LD₅₀ BALB/c-Ly49H⁺ IFNAR^-/-: 2x10³ pfu; LD₅₀ BALB/c: 2.2x10⁴ pfu; LD₅₀ BALB/c MyD88^-/-: 8x10³ pfu; LD₅₀ BALB/c IFNAR^-/-: 2x10³ pfu. (B) Splenic viral titers were measured at d1.5, 3 and 6 post-infection. Data (mean±SEM) are shown from 2 to 4 pooled independent experiments, each with 2 to 3 mice per group.</p

Other inflammatory markers of the IDO1^−/− caput epididymidis.

<p><b>A:</b> Quantitative RT-PCR estimations of <i>COX1</i> and <i>COX2</i> mRNA accumulation in caput epididymidis samples from <i>wt</i> (black bars) and <i>IDO1^−/−</i> (grey bars) male mice. For quantification of transcripts, the relative method was used to calculate mRNA levels relative to <i>Cyclophilin B</i> standard. WT levels were set as 1 in the Y-axis. Mean +/− SEM; n = 12. *<i>P</i>≤0.05; **<i>P</i>≤0.01. <b>B:</b> Schematic representation of the omega 6- (ω6) and omega 3-derived (ω3) fatty acid (FA) intermediates (DGLA, Dihomo-gamma-linolenic acid; AA, Arachidonic acid; EPA, Eicosapentaenoic acid and DHA, Docosahexaenoic acid) precursors of the eicosanoids derivatives including prostaglandins, leucotrienes and thromboxanes. Bracketed numbers given above each FA species indicate difference recorded in the representation of these FA in caput epididymidis extracts of <i>IDO1^−/−</i> animals versus <i>wt</i> (n = 6). <b>C:</b> Histograms illustrate the recorded differences in sphingolipid intermediates (sphingomyelin and ceramides) concentration in caput epididymidis extracts of <i>wt</i> (black bars) and <i>IDO1^−/−</i> (grey bars) from 6 month-old animals. Cholesterol level was taken as a reference to show that the transgenic animals do not present a general disruption in their epididymal lipidic profile.</p

Chemokine profiles of the epididymis and plasma.

<p>Histograms show the levels of the inflammatory chemokines CCL1 (TCA-3, T-cell activation 3), CCL2 (MCP1, Monocyte chemoattractant protein 1), CCL3 (MIP-1α, Macrophage inflammatory protein 1), CCL5 (RANTES, regulated on activation normal T cell expressed and secreted), CCL11 (Eotaxin 1), CCL24 (Eotaxin 2), CCL25 (TECK, Thymus-Expressed Chemokine), CXCL5 (LIX, Lipopolysaccharide-induced CXC-chemokine), CXCL9 (MIG, Monokine induced by gamma interferon), CXCL11 (I-TAC, Interferon-inducible T-cell alpha chemoattractant), CXCL13 (BCA-1, B-cell attracting chemokine 1) and CX3CL1 (Neurotactin) in caput epididymidis extracts (<b>A</b>) and plasma (<b>B</b>) from 6 month-old <i>wt</i> and <i>IDO1−/−</i> animals. *<i>P</i>≤0.05; **<i>P</i>≤0.01.</p

Analysis of the inflammatory status of the epididymis.

<p><b>A:</b> KYN:TRP ratios in caput epididymis and blood plasma, respectively, of 6 month-old <i>wt</i> and <i>IDO1−/−</i> animals. The Y-axis is a semi logarithmic representation in µmole/mmole. <b>B:</b> Histograms show the levels of INF-γ and TNF-α in caput epididymidis extracts and plasma from 6 month-old <i>wt</i> and <i>IDO1−/−</i> animals. <b>C:</b> Histogram shows the levels of the soluble TNF receptors (sTnfRI and sTnfRII) in caput epididymidis extracts from 6 month-old <i>wt</i> and <i>IDO1−/−</i> animals. *<i>P</i>≤0.05, **<i>P</i>≤0.01, ***<i>P</i>≤0.001.</p

Monitoring of the activation of the TGFß/BMP pathway in caput epididymis extracts.

<p>Bar graphs showing the levels of Smad3 and Smad1-5 proteins and their phosphorylated counterparts phosphoSmad3 and phosphoSmad1-5 upon activation in caput epididymidis extracts from <i>wt</i> and <i>IDO1^−/−</i> mice at 6 months of age. Bar graphs display means ± SEM using GAPDH as an internal standard for quantification (n = 4 for Smad3, and n = 8 for Smad1-5; *<i>P</i>≤0.05; **p<0.01).</p

Interleukin profile of the epididymis.

<p>Histograms show the levels of the interleukins IL-1ß, IL-2, IL-3, IL-4, IL-6, IL-9, IL-10, IL-12p70, IL-12p40p70, IL-13, and Il-17 in caput epididymidis extracts (<b>A</b>) and plasma (<b>B</b>) from 6 month-old <i>wt</i> and <i>IDO1−/−</i> animals. *<i>P</i>≤0.05; **<i>P</i>≤0.01.</p