<i>P. chabaudi</i> infection induces IFNB production in pDCs and RPMs.

<p>(A) No splenocytes are YFP⁺ in mock-infected samples, but some splenocytes become YFP⁺24 h after <i>P. chabaudi</i> infection of C57BL/6 <i>Ifnb</i> reporter mice. 2.5×10⁶ events are depicted in each dot plot. (B) pDCs and RPMs constitute over 90% of YFP⁺ events in C57BL/6 mice. (C) Both pDCs and RPMs contribute to splenic IFNB production in 129Sv mice. pDCs were depleted with a single 500 µg dose of anti-mPDCA1 antibody 18 h before infection with <i>P. chabaudi</i>. Grey dots represent individual mice, with horizontal bars representing the mean (B) or geometric mean (C).</p

Mice lacking RPMs exhibit wild type infection kinetics.

<p>(A) Parasitemia courses in 129Sv <i>SpiC^+/−</i> (<i>n</i> = 4) and <i>SpiC^−/−</i> (<i>n</i> = 5) mice are represented as geometric means with standard deviations and Mann-Whitney <i>p</i>-value. (B) Ly6c^lo monocyte (CD11b⁺ F4/80⁺ Ly6g⁻ SSC^lo Ly6c^lo) frequencies in blood and spleen of 129Sv <i>SpiC^+/−</i> (white bars) and <i>SpiC^−/−</i> mice (black bars) during the course of infection. Days depicted in blue and orange represent a 1.5-fold decrease or increase, respectively, in Ly6c^lo monocyte frequencies in blood and spleen of mice infected with <i>P. chabaudi</i>. (C) Ly6c^lo monocyte frequencies on day 12 post-infection. Means are presented with standard errors; <i>p</i>-values represent a two-tailed <i>t</i>-test assuming unequal variances. Data represent three independent experiments (<i>n</i> = 6–7 mice per group total).</p

T1IFN and IFNG signaling redundantly regulate early gene expression responses to <i>P. chabaudi</i> infection.

<p>A representative set of ISG is shown for the gene expression response in whole blood from animals infected for 24 h with <i>P. chabaudi</i> in C57BL/6 knockout mice. Each column represents an individual mouse.</p

Molecular requirements for splenic T1IFN transcriptional induction.

<p>(A) <i>Tlr9</i> and <i>Myd88</i> are required for full transcriptional induction of T1IFNs in spleens of <i>P. chabaudi</i>-infected C57Bl/6 mice. Grey dots represent the means of independent experiments using 4–6 total mice, with T1IFN fold mRNA induction in knockout mice represented as a percentage of induction in wild type animals. Black bars represent means; asterisks represent <i>p</i><0.05 as compared to wild type induction in a two-tailed Student’s <i>t</i>-test assuming unequal variances. (B) <i>Irf7</i> is required for full T1IFN induction, and <i>Irf3</i> is required for full <i>Ifna</i> but not <i>Ifnb</i> induction. (C) <i>Ifnar1</i> is required for full <i>Ifna</i> induction but dispensable for <i>Ifnb</i> induction, whereas <i>Ifngr1</i> is dispensable for all T1IFN induction.</p

Cellular requirements for splenic T1IFN transcriptional induction.

<p>(A) RPMs, but not other macrophage or dendritic cell subsets, induce <i>Ifna</i> and <i>Ifnb</i> at 24 h post-infection with <i>P. chabaudi</i> as detected by qRT-PCR in C57BL/6 mice. Fold mRNA induction represents fold induction of transcript in infected versus mock-infected normalized to beta-actin. Grey dots represent independent experiments conducted on different days; black bars denote the geometric means of the fold inductions. (B) pDCs are not required for splenic <i>Ifna</i> or <i>Ifnb</i> transcriptional induction in response to <i>P. chabaudi</i> in C57BL/6 mice. (C) Genetic deletion of RPMs in 129Sv mice results in diminished T1IFN transcriptional induction. (D) Genetic deletion of <i>Myd88</i> from dendritic cells does not impact transcriptional induction of T1IFNs in spleens of C57BL/6 mice. (E) Genetic deletion of <i>Myd88</i> from macrophages/neutrophils decreases transcriptional induction of T1IFNs by an order of magnitude in C57Bl/6 mice. Grey bars represent geometric means with 95% confidence intervals. Asterisks represent <i>p</i><0.05 in a Student’s <i>t</i>-test against control samples.</p

T1IFNs contribute to control of <i>P. chabaudi</i> infection.

<p>Infected mice were monitored for parasitemia by thin blood smear and survival. Wild type C57BL/6 and congenic knockout mice were infected and monitored for percent parasitemia (<i>n</i> = 5 per strain), which are represented as geometric means with standard deviations and Mann-Whitney <i>p</i>-value. A representative experiment of two is shown. Crosses indicate deaths due to parasitemia.</p

T1IFNs are produced during <i>P. chabaudi</i> infection.

<p>(A) Kinetics of early <i>Ifna, Ifnb</i>, and <i>Ifng</i> transcription using whole spleen qRT-PCR. Fold mRNA induction represents the ratio of transcript in infected over mock-infected C57BL/6 mice. (B) Reproducibility of T1IFN transcript induction as detected by whole spleen qRT-PCR. Each point represents an independent experiment with 4–6 animals, with horizontal bars displaying the geometric mean. (C) Plasma IFNA and IFNB at 24 h post-infection. Data are combined from two independent experiments with each point representing one animal. N.D. = not detected (<i>n</i> = 8).</p

Flagellin and CpG ODN induce robust innate inflammatory infiltrates in the lung.

<p>(<b>A</b>), (<b>B</b>) Neutrophil accumulation in the airways one day after a single i.n. administration (d1) of OVA (O) or OVA plus flagellin (1 μg) (OFla) in wildtype, <i>Tlr5</i>^<i>-/-</i><i>Tlr11</i>^<i>-/-</i>, and <i>Nlrc4</i>^<i>-/-</i> mice (<b>A</b>) or in wildtype, <i>Tlr5</i>^<i>-/-</i><i>Tlr11</i>^<i>-/-</i> and <i>Tlr4</i>^<i>-/-</i> mice (<b>B</b>), as assessed by flow cytometry of the cells in the BAL fluid. (<b>C</b>) Cellular composition of the innate inflammatory infiltrate in the lung one day after the third i.n. sensitization (d3) with OVA, OVA plus flagellin (1 μg), or OVA plus CpG ODN (3 μg) (OCpG). Data in (<b>A</b>) contain 4 mice per group and are representative of two independent experiments, data in (<b>B</b>) contain 4 mice per group and are representative of two independent experiments, and data in (<b>C</b>) contain 3–4 mice per group and are representative of three independent experiments. Error bars indicate mean +SD. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 using one-way anova with Bonferroni post-test. In (<b>A</b>) and (<b>B</b>), all groups of OVA-treated mice have statistically significant different values compared to OVA plus flagellin-treated wild-type mice (<b>A</b> and <b>B</b>), <i>Nlrc4</i>^<i>-/-</i> (<b>A</b>), and <i>Tlr4</i>^<i>-/-</i> mice (<b>B</b>) (*** P ≤ 0.001 for all comparisons; not indicated on the panel).</p

Migratory DCs require MyD88 signaling to respond normally to i.n. exposure to flagellin or CpG ODN.

<p>Expression of activation markers on migratory DCs in the lung-draining, mediastinal LNs of <i>Myd88</i>^<i>fl/fl</i> (<i>M</i>^<i>F</i>) and <i>Myd88</i>^<i>fl/fl</i> <i>CD11c-Cre</i> (<i>M</i>^<i>F</i> <i>CD11c-Cre</i>) mice one day after i.n. administration (d1) of OVA-AF647 or OVA-AF647 plus TLR ligand. (<b>A</b>) Migratory DCs were gated as CD11c⁺I-A^b(hi), then gated according to OVA-AF647 expression. (<b>B</b>) and (<b>C</b>) Comparison of different activation markers on migratory DCs between <i>M</i>^<i>F</i> and <i>M</i>^<i>F</i> <i>CD11c-Cre</i> mice treated i.n with OVA-AF647, OVA-AF647 plus flagellin (1 μg), or OVA-AF647 plus CpG (0.75 or 3 μg). (<b>B</b>) Representative histograms of different activation markers on migratory DCs that did take up OVA-AF647. (<b>C</b>) Level of expression (MFI) of activation markers on migratory DCs that did (OVA⁺) or did not (OVA^-) take up fluorescent OVA. (<b>D</b>) and (<b>E</b>) Comparison of different activation markers on migratory DCs between <i>M</i>^<i>F</i> and <i>M</i>^<i>F</i> <i>CD11c-Cre</i> treated i.n with OVA-AF647 or OVA-AF647 plus CpG ODN (0.75 μg). (<b>D</b>) Representative histograms of different activation markers on migratory DCs that did take up OVA-AF647. (<b>E</b>) Level of expression (MFI) of activation markers on migratory DCs that did (OVA⁺) or did not (OVA^-) take up fluorescent OVA. Data in (<b>B</b>) and (<b>C</b>) contain 3–4 mice per group and are representative of 3 independent experiments using OVA-AF647 and a fourth independent experiment using non-fluorescent OVA. Data in (<b>D</b>) and (<b>E</b>) contain 4–6 mice per group and are representative of two independent experiments. Negative control histograms (solid light gray) were from CD11c^-I-A^b- cells. Each circle represents an individual mouse. Error bars indicate mean +SD. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 using one-way anova with Bonferroni post-test.</p

Flagellin, but not CpG ODN, promotes development of IL-4-producing CD4 T cells in the draining LN.

<p>4get/KN2 reporter mice were administered OVA, OVA plus flagellin (1 μg), or OVA plus CpG (0.75 μg) i.n. on d0, 1 and 2. In addition, to block IL-12 action in some mice, mice were given anti-IL-12 p40 or control antibody (rat IgG2a) twice, one day before initial sensitization (700 μg i.p.) and again on d2 (300 μg i.p.). On d6, expression of IL-4 reporters (GFP⁺hCD2⁺) by CD4 T cells was examined in the mediastinal LN. (<b>A</b>) Gating strategy of CD4 T cells (CD4⁺), activated CD4 T cells (CD4⁺CD44^hiB220^-CD62L^-), and T_FH cells (CD4⁺CD44^hiB220^-CD62L^-PD-1⁺CXCR5⁺). (<b>B</b>) Numbers of CD4 T cells, percentages and numbers of GFP⁺IL-4⁺(hCD2⁺) CD4 T cells. (<b>C</b>) Percentages and numbers of activated CD4 T cells, percentages and numbers of T_FH cells, and percentages and numbers of GFP⁺IL-4⁺(hCD2⁺) T_FH. Data are pooled from two independent experiments with combined totals of 8 mice per group. Each circle represents one individual mouse. Error bars indicate mean +SD. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 using one-way anova with Bonferroni post-test.</p