The Non-receptor Tyrosine Kinase Tec Controls Assembly and Activity of the Noncanonical Caspase-8 Inflammasome

<div><p>Tec family kinases are intracellular non-receptor tyrosine kinases implicated in numerous functions, including T cell and B cell regulation. However, a role in microbial pathogenesis has not been described. Here, we identified Tec kinase as a novel key mediator of the inflammatory immune response in macrophages invaded by the human fungal pathogen <i>C. albicans</i>. Tec is required for both activation and assembly of the noncanonical caspase-8, but not of the caspase-1 inflammasome, during infections with fungal but not bacterial pathogens, triggering the antifungal response through IL-1β. Furthermore, we identify dectin-1 as the pathogen recognition receptor being required for Syk-dependent Tec activation. Hence, Tec is a novel innate-specific inflammatory kinase, whose genetic ablation or inhibition by small molecule drugs strongly protects mice from fungal sepsis. These data demonstrate a therapeutic potential for Tec kinase inhibition to combat invasive microbial infections by attenuating the host inflammatory response.</p></div

MAZR is not essential for the differentiation of BM-derived mast cells.

<div><p>(A) Diagram shows qRTPCR analysis of <i>Mazr</i> expression in thymus, CD4⁺ and CD8⁺ T cells and in IgE-primed BM-derived mast cells (BMMC). Expression levels are normalized to <i>Hprt</i> expression and levels in thymocytes were set as 1 (100%). Columns represent a summary of three independent samples. Mean ± SEM is shown.</p> <p>(B) Histograms depict expression of cell surface markers on <i>Mazr</i>^F/F and <i>Mazr</i>^<i>F/F</i><i>Vav-iCre</i> BMMC (after 5 weeks of culture). Filled gray areas are isotype control stainings. Data are representative of three independent experiments.</p> <p>(C) Flow cytometric analysis showing up-regulation of FcεRI levels in <i>Mazr</i>^F/F and <i>Mazr</i>^<i>F/F</i><i>Vav-iCre</i> BMMC. Filled gray areas are isotype control stainings. The solid black line shows the levels of cell-surface bound IgE after 15 min incubation. The dotted line shows the levels of cell-surface bound IgE after overnight priming. Data are representative of three independent experiments. </p> <p>(D) Toluidine blue staining of 5 week-cultured <i>Mazr</i>^F/F and <i>Mazr</i>^<i>F/F</i><i>Vav-iCre</i> BMMC prepared by a cytospin. Magnification 20x.</p></div

Minor defects in early and late mast cell effector functions in the absence of MAZR.

<div><p>(A) Diagram shows qRTPCR analysis of <i>Mazr</i> expression in resting anti-TNP IgE-primed BMMCs and in BMMCs activated for the indicated time points with TNP. Expression levels are normalized to <i>Hprt</i> expression and levels in IgE-primed non-activated mast cells were set as 1 (100%). Data show summary of three samples analyzed. Mean ± SEM is shown.</p> <p>(B) Plasma histamine levels in a systemic anaphylaxis model are shown. <i>Mazr</i>^F/F and <i>Mazr</i>^<i>F/F</i><i>Vav-iCre</i> mice were primed (i.v.) with anti-TNP IgE, challenged 24 hours later by i.v. injection of TNP or PBS. Serum was collected 2 minutes post-injection and histamine levels were determined by ELISA, n=7.</p> <p>(C) Absorbance (OD) of Evans Blue dye extravasated in a passive cutaneous anaphylaxis model from the ears of <i>Mazr</i>^F/F and <i>Mazr</i>^<i>F/F</i><i>Vav-iCre</i> mice is shown. Mice were injected with PBS and anti-TNP IgE into left and right ear, respectively, and 24 hours later mice were challenged by i.v. injection with TNP/Evans Blue dye. Extravasation of Evans Blue dye in the ear was measured 4 hours later. Diagram shows summary of 9 mice. Mean ± SEM is shown.</p> <p>(D) Anti-TNP-IgE-primed <i>Mazr</i>^F/F and <i>Mazr</i>^<i>F/F</i><i>Vav-iCre</i> BMMCs were activated for 10 min with TNP or PMA/ionomycin. β-Hexosaminidase release levels of <i>Mazr</i>^<i>F/F</i> BMMCs were set to 1 (n=10).</p> <p>(E) Anti-TNP-IgE-primed <i>Mazr</i>^F/F and <i>Mazr</i>^<i>F/F</i><i>Vav-iCre</i> BMMCs were activated for 60 min with TNP. LTB₄ levels were determined by ELISA. Mean ± SEM is shown. (n=4).</p> <p>(F) Flow cytometric analysis of calcium flux in anti-TNP-IgE-primed <i>Mazr</i>^F/F and <i>Mazr</i>^<i>F/F</i><i>Vav-iCre</i> BMMCs that have been activated with TNP. Data are representative of 3 independent experiments. </p> <p>(G) Cytokine production of anti-TNP-IgE-primed <i>Mazr</i>^F/F and <i>Mazr</i>^<i>F/F</i><i>Vav-iCre</i> BMMCs that were activated by plate-bound TNP for 24 hours (at least 5 independent mast cell batches were analyzed). Cytokine production of <i>Mazr</i>^<i>F/F</i> BMMCs was set to 1.</p></div

Chemical-genetic inhibition of Tec rescues mice from fatal fungal sepsis.

<p>(<b>a</b>) Survival of mice after intraperitoneal infection with 5.10⁷ CfUs of <i>C. albicans</i> and oral treatment with PCI-32765 with daily doses as indicated; treatment was stopped after 5 days; for analysis of mouse survival curves Log-rank (Mantle-Cox) test was used. n = 9 per group. (<b>b</b>) Survival of mice after intraperitoneal infection with 5.10⁷ CfUs of <i>C. albicans</i> and oral treatment with 5 mg/kg bodyweight PCI-32765 with daily doses; for analysis of mouse survival curves Log-rank (Mantle-Cox) test was used. n = 6 per group. (<b>c</b>) Survival of mice after intraperitoneal infection with 5.10⁷ CfUs of <i>C. albicans</i> and oral treatment with 5 mg/kg bodyweight PCI-32765 with daily doses; treatment was stopped after 9 days; for analysis of mouse survival curves Log-rank (Mantle-Cox) test was used. n = 6 per group.</p

Gene expression analysis of non-activated MAZR-deficient BMMCs.

<div><p>(A) Gene expression profiles from IgE-primed (but non-activated) <i>Mazr</i>^F/F and <i>Mazr</i>^<i>F/F</i><i>Vav-iCre</i> BMMC were determined using Agilent arrays. Data were analyzed using GeneSpring software as described in materials and methods. The scatter plot indicates the 128 genes that are dysregulated in the absence of MAZR (log₂ expression levels; ≥2 fold-difference, P≤0.1). Numbers at the upper-left or lower-right corners show the number of genes specifically expressed in <i>Mazr</i>^<i>F/F</i><i>Vav-iCre</i> (Y-axis) and <i>Mazr</i>^F/F (X-axis) BMMCs. The highlighted genes are selected from the top-ten hits with the largest-fold difference between <i>Mazr</i>^F/F and <i>Mazr</i>^<i>F/F</i><i>Vav-iCre</i> BMMC.</p> <p>(B) qRTPCR analysis of genes selected from the microarray experiments. Graphs represent relative expression levels of genes up- and down-regulated in the absence of MAZR (normalized to <i>Hprt</i>). Expression levels in <i>Mazr</i>^F/F samples were set to 1. Mean ± SEM is shown. Data are means of the results from duplicated qRTPCRs of one batch of mast cells. The IgE-primed <i>Mazr</i>^F/F and <i>Mazr</i>^<i>F/F</i><i>Vav-iCre</i> BMMC samples were from a different batch compared to the ones used for probing with Agilent arrays.</p></div

Lack of Tec impairs the inflammatory response to fungal pathogens.

<p>(<b>a</b>) Immunoblot analysis of Tec and (<b>b</b>) qPCR analysis of Tec expression after stimulating BMMs with <i>C. albicans</i> for 120 min; results are normalized to GAPDH (glyceraldehyde phosphate dehydrogenase). (<b>a</b>) Immunoblot of activated Tec in cell lysates after stimulation with <i>C. albicans</i>; lysates were enriched for phospho-proteins. (<b>b</b>) Detection of reactive oxygen species from BMMs after <i>C. albicans</i> challenge for 120 Min using luminol (ROS from unstimulated cells was subtracted). (<b>c</b>) qPCR analysis of cytokine response after 120 Min without (-) or with stimulation with <i>C. albicans</i> (Ca); results are normalized to GAPDH. (<b>d</b>) ELISA for cytokines in supernatants of BMMs with or without (-) <i>C. albicans</i> (Ca) stimulation. (<b>e</b>) Rate of phagocytosis after 45 Min of incubation with <i>C. albicans</i> (Ca). (<b>f</b>) Immunoblotting of p-IκBα and p-NF-κB p65 activation over the time course of <i>C. albicans</i> infection in BMMs. Data are representative of at least two (<b>a</b>–<b>c</b>, <b>g</b>) or three (<b>d</b>–<b>f</b>) independent experiments. Mean and SD are shown.</p

Reduced mast cell numbers <i>in vitro</i> but normal mast cell homeostasis <i>in vivo</i>.

<div><p>(A) Diagram showing the cumulative numbers of c-kit⁺FcεRI⁺<i>Mazr</i>^F/F and <i>Mazr</i>^<i>F/F</i><i>Vav-iCre</i> BMMC over the course of 5 weeks of culture. Cells were counted by CASY counter, then percentages of c-kit⁺FcεRI⁺ BMMC among PI-negative cells (= alive) was determined by flow cytometry. The summary of three independent experiments with a total of 6 independent cell batches is shown. Mean ± SEM is shown.</p> <p>(B) Number of PI-negative (= alive) <i>Mazr</i>^F/F and <i>Mazr</i>^<i>F/F</i><i>Vav-iCre</i> BMMC during 5 days of IL-3 starvation. The summary of 3 experiments is shown. Mean ± SEM is shown.</p> <p>(C) Toluidine blue staining of paraffin-embedded 5 µm ear sections of <i>Mazr</i>^F/F and <i>Mazr</i>^<i>F/F</i><i>Vav-iCre</i> mice showing mast cells in pink/purple color (examples indicated by arrowheads). Diagram at the right indicates mean ± SEM of mast cell number per field of view (fov) calculated over 10 individual sections per ear (n=4). Magnification 20x.</p> <p>(D) Percentage of EYFP⁺c-kit⁺FcεRI⁺ mast cells from peritoneal lavage of wild-type (Mazr^F/+Rosa26^+/EYFPMcpt5Cre) and mast cell-specific MAZR-null (Mazr^F/FRosa26^+/EYFPMcpt5Cre) mice (n=8 and 9, respectively).</p></div

Tec is required for the assembly of the caspase-8 inflammasome.

<p>(<b>a</b>) Caspase-8 activity after 60 Min of stimulation with <i>C. albicans</i> of cells left untreated, knockdown of a non-target (nTG; 25 nM) or respective siRNA knock down (25 nM) after 72 hrs of incubation; chemiluminenscence of unstimulated cells and <i>C. albicans</i> only was subtracted. (<b>b</b>–<b>e</b>) Immunoblot analysis of CARD9, Bcl-10, MALT1, ASC and caspase-8 (Casp8) after immunoprecipitation (IP) with antibodies against CARD9 (<b>b</b>), MALT1 (<b>c</b>), ASC (<b>d</b>) and caspase-8 (<b>e</b>) from whole-cell lysates of BMMs left unstimulated (-) or stimulated with <i>C. albicans</i> for 60 Min. Data are representative of two independent experiments for each IP. (<b>f</b>) Immunoblot analysis of p-Src, p-Syk, p-PLCγ2, p-PKCδ and p-RAF1 during the course of BMM infection with <i>C. albicans</i>. (<b>g</b>) <i>In vitro</i> kinase assay; Tec was immunoprecipitated from unstimulated BMMs and incubated with recombinant active Syk, BSA (70 ng each) and adenosine triphosphate (ATP, 200 nM) for 30 Min at 30°C; active phosphorylated Tec was detected with α-p-Tyr antibodies. (<b>h</b>) <i>In vitro</i> kinase assay; PLCγ2 was immunoprecipitated from unstimulated BMMs and incubated with active Tec, BSA (70 ng each) and adenosine triphosphate (ATP, 200 nM) for 30 Min at 30°C; active PLCγ2 was detected with α-p-PLCγ2 antibody. (<b>i</b>) Immunoblot of activated Tec and p-Src/p-Syk in cell lysates after stimulation with <i>C. albicans</i> and parallel Syk inhibition with R406 (3 µM); lysates were enriched for phospho-proteins. Data are representative of at least two (<b>b</b>–<b>i</b>) or three (<b>a</b>) independent experiments. Mean and SD are shown (<b>a</b>).</p

Tec regulates fungal virulence <i>in vivo</i>.

<p>(<b>a</b>) Caspase-8 activity of total murine peritoneal lavage cells obtained after intraperitoneal infection (<i>i.p.</i>) with 5.10⁶ CfUs of <i>C. albicans</i> after indicated times of infection or control lavage cells from uninfected mice; n = 3 per genotype and time point (<b>b</b>) Caspase-1 activity of total murine peritoneal cells upon <i>i.p.</i> infection with 5.10⁶ CfUs of <i>C. albicans</i> after indicated times of infection or cells from uninfected mice; n = 3 per genotype and time point (<b>c</b>) Intracellular staining of active caspase-8 in neutrophils (CD11b⁺Ly6C⁺Ly6G⁺) and macrophages (CD11b⁺F4/80⁺) in peritoneal lavage cells obtained after i.p. infection with 5.10⁶ CfUs of <i>C. albicans</i> after 24 h. (<b>d</b>) Mice were infected <i>i.p.</i> with 3% brewer thioglykolate medium for 4 days; lavage cells were collected; total caspase-8 activity was measured after 60 Min of stimulation with <i>C. albicans</i> (Ca); chemiluminenscence of unstimulated cells or <i>C. albicans</i> only was subtracted; n = 4 per genotype and time point (<b>e</b>) Polymorph-nuclear neutrophils were isolated from bone marrow of respective mice using a Percoll-gradient; Caspase-8 activity was measured after 60 Min of stimulation with <i>C. albicans</i> (Ca); chemiluminenscence of unstimulated cells or <i>C. albicans</i> only was subtracted. (<b>f</b>) qPCR analysis of TNFα and IL-1β of total murine peritoneal cells upon intraperitoneal infection (<i>i.p.</i>) with 5.10⁶ CfUs of <i>C. albicans</i> after indicated time of infection or cells from uninfected mice; results are normalized to those of GAPDH. n = 3 per genotype and time point (<b>g</b>) ELISA of TNFα and IL-1β in lavage of mice upon intraperitoneal infection (<i>i.p.</i>) with 5.10⁶ CfUs of <i>C. albicans</i> after indicated time of infection or from uninfected mice. n = 3 per genotype and time point. Data are representative of at least two (<b>c</b>) three (<b>a,b, e–g</b>) or four (<b>d</b>) independent experiments. Mean and SD are shown.</p

Caspase-8 activity in response to <i>C. albicans</i> requires Tec in BMMs.

<p>(<b>a</b>) Caspase-1 activity over the course of infection with <i>C. albicans</i>; absorbence of unstimulated cells and <i>C. albicans</i> only was subtracted. (<b>b</b>) Caspase-8 activity over the course of infection with <i>C. albicans</i>; chemiluminenscence of unstimulated cells and <i>C. albicans</i> only was subtracted. (<b>c</b>) Immunoblot analysis of full-length or active (p20) caspase-1 and full-length and active (p10) caspase-8 during the course of BMM infection with <i>C. albicans</i>. (<b>d</b>) ELISA of IL-1β in supernatants of BMMs after stimulation with <i>C. albicans</i> only (Ca) or with dimethylsulfoxide (DMSO), Casp1 inhibitor (Casp1 Inh; 5 mM) or Casp8 inhibitor (Casp8 Inh; 5 mM) and Ca or left unstimulated (-). (<b>e</b>) ELISA of pro-IL-1β in supernatants of BMMs after stimulation with <i>C. albicans</i> (Ca) or left unstimulated (-). Data are representative of at least two (<b>c</b>), three (<b>d,e</b>) or five (<b>a</b>,<b>b</b>) independent experiments. Mean and SD are shown.</p