Mice with targeted deletion of Syk in CD11c⁺ cells show increased susceptibility to systemic candidiasis.

<p>(A) Control and CD11cΔSyk mice were infected with 5×10⁴ CFU of <i>C. albicans</i> intravenously. Survival data are presented as a Kaplan-Meier plot with a log rank test used to compare susceptibility between the two groups. Data are pooled from two independent experiments. (B) 4× and 20× magnification of Periodic Acid Schiff (PAS) or Hematoxilyn and Eosin (H&E) stained kidney sections collected at the endpoint of the experiment shown in (A). Scale bars are 50 µm (4× magnification) and 250 µm (20× magnification), respectively. (C–D) control Syk, CD11cΔSyk, control MyD88, CD11cΔMyD88 and MyD88 KO mice (C) and control Clec9a, Clec9aΔSyk and Clec9a(egfp/egfp) mice (D) were infected with 2×10⁵ CFU of <i>C. albicans</i> intravenously. Kidneys were removed 2 days post-infection and analyzed for fungal burden. CFU are calculated per gram of kidney. Data shown are mean +/− SEM from pooled from two to six independent experiments with each symbol representing an individual mouse with statistical significance of any differences determined using a 1-way ANOVA with Tukey post-test analysis.</p

Transfer of <i>in vivo</i> activated NK cells leads to restoration of fungal control in CD11cΔSyk mice.

<p>(A) Fungal burden in kidneys from control and CD11cΔSyk mice, as well as CD11cΔSyk mice receiving adoptively transferred NK cell-enriched splenocytes from either naïve or day 1 infected wild type mice. Data are mean +/− SEM pooled from two independent experiments with each symbol representing an individual mouse. (B) Global geometric mean of CD11b expression by infiltrating neutrophils from the mice shown in (A). Data are mean +/− SEM from two independent experiments with each symbol representing neutrophils from an individual mouse. (C) Neutrophils were isolated from the blood of the mice shown in (A) and co-cultured with <i>C. albicans</i> to assess candidacidal activity. Data are mean +/− SEM from two independent experiments with each symbol representing an individual mouse. (D) Fungal burden of kidneys from control and CD11cΔSyk mice as well as CD11cΔSyk mice that received sorted splenic NK cells or unsorted total splenocytes isolated from 16 h or 48 h infected control mice. Data are mean +/− SEM from two independent experiments with each symbol representing an individual mouse. (E) Neutrophils identified as live, CD45.2⁺, Ly-6G⁺ and CD11b⁺ cells, from the mice shown in (D) were assessed for their expression of CD11b. Representative FACS plots are shown. The percentage of CD11b^hi and CD11b^lo cells is indicated. Statistical significance of any differences for A, B and C was determined using a 1-way ANOVA with Tukey post-test analysis. Statistical significance of any differences for D was determined by 2-tailed <i>t</i> test. NS, not significant.</p

Selective loss of GM-CSF in the kidneys of CD11cΔSyk mice and restoration of fungal control by exogenous GM-CSF administration.

<p>(A) Kidneys were removed from naïve mice or mice 16 h post-infection mice and assessed using a Proteome profiler. Data show relative pixel density of duplicate blots for GM-CSF and G-CSF protein expression changes following infection. (B) Control, CD11cΔSyk and MyD88 KO mice were given PBS or GM-CSF at the time of <i>C. albicans</i> infection as indicated and a second dose 24 h later. Fungal burden was assessed 2 days post-infection. Data are mean +/− SEM from two pooled experiments with each symbol representing an individual mouse. Statistical significance of any differences was determined by 2-tailed <i>t</i> test. NS, not significant.</p

Increased infiltration of defective neutrophils in the kidneys of CD11cΔSyk mice.

<p>Control and CD11cΔSyk mice were infected with 2×10⁵ CFU of <i>C. albicans</i> intravenously. Kidneys were removed from naïve mice or mice 2 days post-infection and leukocytes were surface stained for CD11b, F4/80, MHC-II and Ly-6G. (A) Percentage and total number of Ly6G⁺ CD11b⁺ kidney neutrophils. Data are mean +/− SEM from four pooled independent experiments with each data point representing an individual mouse. (B) Representative expression of CD11b and CD11a on infiltrating kidney neutrophils of day 2 infected control and CD11cΔSyk mice. (C) Global geometric mean of CD11b expression on infiltrating kidney neutrophils normalized against control naïve mice. Data are mean +/− SEM from four independent experiments with each data point representing an individual mouse. (D) Neutrophils from day 2 infected mice were permeabilized and stained for MPO. Data shown are global geometric mean of MPO signal on infiltrating kidney neutrophils normalized against control mice. Data are combined from two independent experiments with each data point representing an individual mouse. (E) Control and CD11cΔSyk were infected with 2×10⁵ CFU of <i>C. albicans</i>-GFP intravenously. Kidneys were removed 2 days post-infection and analyzed for GFP expression. Representative staining profiles of total CD45.2⁺ kidney cells (top panel) and CD45.2⁺ Ly-6G⁺ neutrophils (bottom panel). Boxes indicate the percentages of GFP⁺ neutrophils and the mean +/− SEM from three independent experiments with number (n) of mice indicated. (F) GFP⁺ and GFP⁻ neutrophils (CD11b⁺ Ly-6G⁺ CD11c⁻ F4/80⁻) were sorted, lysed in water and plated to determining presence of viable fungi. Data are combined from two independent experiments with each data point representing cells sorted from an individual mouse. (G) Neutrophils were sorted from naïve BM or day 2 infected kidneys of control and CD11cΔSyk mice and incubated with <i>C. albicans</i> (10∶1) for 1 h at 37°C. The survival of fungi was then assessed. Data are mean +/− SEM from three independent experiments with each data point representing cells sorted from an individual mouse. Statistical significance of any differences for A, C, D and G was determined by 2-tailed <i>t</i> test. Whilst a Kruskal-Wallis with Dunn's multiple comparison test was undertaken for F. NS, not significant.</p

Model for Syk-mediated recognition of fungal particles by DCs in control of systemic candidiasis.

<p>Upon systemic infection, recognition of <i>C. albicans</i> by kidney DCs via Syk-coupled PRRs leads to production of IL-23p19. DC-derived IL-23p19 acts on NK cells, causing them to produce GM-CSF, which in turn acts on neutrophils infiltrating the kidney to promote and sustain fungicidal activity. Loss of Syk in DCs prevents production of IL-23p19 leading to failure of NK cells to provide GM-CSF-dependent help to neutrophils. Impaired killing capacity of the latter leads to loss of control of fungal burden.</p

Defect in IL-23p19 production by Syk-deficient kidney DCs underlies decreased NK cell GM-CSF-mediated control of <i>C. albicans</i>.

<p>(A) Representative staining of naïve control kidney NK cells (CD45.2⁺ NK1.1⁺ CD3⁻) and T cells (CD45.2⁺ CD3⁺) with anti-IL-23R vs. isotype-matched irrelevant specificity control (B) Percentage and total number of kidney NK cells expressing IL-23R in control and CD11cΔSyk mice that were either uninfected or infected 16 h earlier. Data are mean +/− SEM from three independent experiments with each symbol representing an individual mouse. (C) CD11c⁺ MHC-II⁺ cells were purified by cell sorting from the kidneys of naïve or 16 h post-infection control and CD11cΔSyk mice. RNA was extracted and qRT-PCR performed to detect <i>il23a transcripts</i>. Data shown are mean +/− SEM from two independent experiments with five biological replicates. n.d., not detected. (D) NK cells were purified by cell sorting from the kidneys of naïve and 16 h post-infection control, CD11cΔSyk and IL-23p19 KO mice. RNA was extracted and qRT-PCR performed to detect levels of <i>Csf2</i> and <i>Ifng</i> transcripts. Data shown are mean +/− SEM from two independent experiments with each symbol representing an individual mouse. (E) NK cells were sorted from the spleen and kidney of 16 h-infected control, CD11cΔSyk and IL-23p19KO mice. Cells were cultured overnight and supernatants were collected and assessed for GM-CSF protein content by ELISA. Data shown are mean +/− SEM from two independent experiments with each symbol representing data from an individual animal. (F) Splenic NK cells were sorted from naïve control mice and stimulated with medium, recombinant IL-17AF heterodimer or recombinant IL-23 (r-IL-23) overnight. GM-CSF protein in the supernatants was measured by ELISA. Data shown are mean +/− SEM of triplicate wells from one experiment. (G) Splenic NK cells were sorted from naïve control and CD11cΔSyk mice and were stimulated overnight with medium, heat-killed <i>C. albicans</i>, Curdlan, Zymosan or PMA/Ionomycin. GM-CSF accumulation in the supernatants was assessed by ELISA. Data shown are mean +/− SEM of duplicate wells from one out of two independent experiments. (H) Splenic NK cells were sorted from naïve control mice and co-cultured with BMDCs derived from control, CD11cΔSyk or IL-23p19KO mice. Co-cultures were stimulated with medium, heat-killed <i>C. albicans</i> or recombinant IL-23 (r-IL-23) overnight and GM-CSF protein accumulation in the supernatants was assessed by ELISA. Data shown are mean +/− SEM of triplicate wells from one out of two independent experiments. n.d., not detected. (I) Control and IL-23p19 KO mice were infected with 2×10⁵ CFU of <i>C. albicans</i> intravenously. Kidneys were removed 2 days post-infection and analyzed for fungal burden. Data are mean +/− SEM from two independent experiments with each symbol representing an individual animal. (J) IL-23p19 KO mice were given PBS or GM-CSF at the time of <i>C. albicans</i> infection and a second dose 24 h later. Fungal burden was assessed 2 days post-infection. Data are mean +/− SEM pooled from two independent experiments with each symbol representing an individual animal. Statistical significance of any differences for B was determined using a 1-way ANOVA with Tukey post-test analysis. C, I and J were assessed using a 2-tailed <i>t</i> test Whilst a Kruskal-Wallis with Dunn's multiple comparison test was undertaken for D. NS, not significant.</p

GM-CSF-producing NK cells are selectively reduced in infected CD11cΔSyk and Clec9aΔSyk mice.

<p>Control, CD11cΔSyk and Clec9aΔSyk mice were infected with 2×10⁵ CFU of <i>C. albicans</i> intravenously. (A) Percentage and total number of NK cells in the kidney of control and CD11cΔSyk mice that were either uninfected or infected 16 h earlier. (B–C) Enriched leukocyte populations from control and CD11cΔSyk mice (B) and from control and Clec9aΔSyk mice (C) that were either uninfected or infected 16 h earlier were analyzed for GM-CSF and IFN-γ by intracellular staining. The percentage and absolute number of cytokine-positive CD45.2⁺ NK1.1⁺ CD3⁻ cells are shown. Each data point represents an individual mouse with bars indicating mean +/− SEM pooled from two to four independent experiments. Statistical significance of any differences was determined using a 1-way ANOVA with Tukey post-test analysis. NS, not significant.</p

table_1_Clec9a-Mediated Ablation of Conventional Dendritic Cells Suggests a Lymphoid Path to Generating Dendritic Cells In Vivo.xlsx

<p>Conventional dendritic cells (cDCs) are versatile activators of immune responses that develop as part of the myeloid lineage downstream of hematopoietic stem cells. We have recently shown that in mice precursors of cDCs, but not of other leukocytes, are marked by expression of DNGR-1/CLEC9A. To genetically deplete DNGR-1-expressing cDC precursors and their progeny, we crossed Clec9a-Cre mice to Rosa-lox-STOP-lox-diphtheria toxin (DTA) mice. These mice develop signs of age-dependent myeloproliferative disease, as has been observed in other DC-deficient mouse models. However, despite efficient depletion of cDC progenitors in these mice, cells with phenotypic characteristics of cDCs populate the spleen. These cells are functionally and transcriptionally similar to cDCs in wild type control mice but show somatic rearrangements of Ig-heavy chain genes, characteristic of lymphoid origin cells. Our studies reveal a previously unappreciated developmental heterogeneity of cDCs and suggest that the lymphoid lineage can generate cells with features of cDCs when myeloid cDC progenitors are impaired.</p

data_sheet_1_Clec9a-Mediated Ablation of Conventional Dendritic Cells Suggests a Lymphoid Path to Generating Dendritic Cells In Vivo.PDF

<p>Conventional dendritic cells (cDCs) are versatile activators of immune responses that develop as part of the myeloid lineage downstream of hematopoietic stem cells. We have recently shown that in mice precursors of cDCs, but not of other leukocytes, are marked by expression of DNGR-1/CLEC9A. To genetically deplete DNGR-1-expressing cDC precursors and their progeny, we crossed Clec9a-Cre mice to Rosa-lox-STOP-lox-diphtheria toxin (DTA) mice. These mice develop signs of age-dependent myeloproliferative disease, as has been observed in other DC-deficient mouse models. However, despite efficient depletion of cDC progenitors in these mice, cells with phenotypic characteristics of cDCs populate the spleen. These cells are functionally and transcriptionally similar to cDCs in wild type control mice but show somatic rearrangements of Ig-heavy chain genes, characteristic of lymphoid origin cells. Our studies reveal a previously unappreciated developmental heterogeneity of cDCs and suggest that the lymphoid lineage can generate cells with features of cDCs when myeloid cDC progenitors are impaired.</p