The non-receptor protein tyrosine kinases Btk and Tec and their role in macrophages in response to infection

Mutationen in Tec-Kinasen (Tec, Btk, Bmx, Itk und Rlk) sind Ursache schwerer Immundefizienzen bei Menschen und ähnlicher Funktionsstörungen bei Mäusen. Funktionen von Tec-Kinasen bei der Regulation der Entwicklung, Aktivierung und Differenzierung von Lymphozyten wurden gut charakterisiert. Etliche Studien beschreiben Funktionen von Tec-Kinasen auch in myeloiden Zellen wie z. B. Makrophagen. Dennoch gibt es nur wenig Wissen über die Rolle von Tec-Kinasen während Infektionen mit lebenden Pathogenen. Im ersten Teil meiner Ph.D.-Arbeit habe ich die Rolle der Bruton's Tyrosinkinase (Btk) in der Immunantwort auf Listeria monocytogenes (Lm) untersucht. Nach Infektion mit Lm wurde Btk in Knochenmarksmakrophagen (KMM) aktiviert und Btk-/- KMM produzierten mehr Zytokine. Da es keine Unterschiede in der Immunantwort auf Hitze-getötete Lm zwischen Wildtyp (wt) und Btk-/- KMMs gab, deuten die Daten auf eine Rolle für Btk in der durch intrazelluläre Lm induzierten Signaltransduktion. Außerdem waren Btk-/- Mäuse resistenter gegen Lm als wt Mäuse, was mit einer reduzierten Bakteriendichte in Btk-/- Mäusen korrelierte. Zusammengefasst zeigen meine Daten eine wichtige regulatorische Funktion von Btk in Makrophagen während Infektionen mit Lm. Im zweiten Teil meiner Ph.D.-Arbeit habe ich eine komprehensive funktionelle Analyse von Tec-Kinase-defizienten Makrophagen nach TLR-Aktivierung durchgeführt. Dafür habe ich Zytokinproduktion und andere Effektorfunktionen von Btk-/-, Tec-/- und Btk/Tec-doppeldefizienten KMM nach Stimulierung mit TLR-Liganden studiert. Ich konnte zeigen, dass Btk und Tec unterschiedlich benötigt werden und dass sie weiters bei der Phagozytose von bakteriellen Fragmenten eine Rolle spielen. Zusammengefasst stellen die Ergebnisse meiner Dissertationsarbeit neuartige Funktionen für die Tec-Kinasen Btk und Tec in unterschiedlichen Aspekten der Immunantwort von Makrophagen auf Infektionen dar.Mutations in Tec family kinases (TFK) (Tec, Btk, Bmx, Itk and Rlk) can cause severe immunodeficiencies in humans and similar disorders in mice. The functions of TFKs have been well described in the lymphoid lineage where they regulate lymphocyte development, activation and differentiation. Several studies investigated also the function of TFKs in cells of the myeloid lineage such as in macrophages. However, there is little knowledge about the role of TFKs during infections with live pathogens. In the first part of my Ph.D. thesis studies, I investigated the role of Bruton's tyrosine kinase (Btk) in the immune response to Listeria monocytogenes (Lm). In response to Lm, Btk was activated in bone marrow-derived macrophages (BMMs) and Btk-/- BMMs showed enhanced cytokine secretion. Since there was no difference in the response to heat-killed Lm between wild-type (wt) and Btk-/- BMMs, the data suggest a role for Btk in signaling pathways induced by intracellular Lm. Moreover, Btk-/- mice displayed enhanced resistance to Lm compared to wt mice and this correlated with enhanced cytokine levels in the serum and reduced bacterial burden in infected Btk-/- mice. Together, these data suggest an important regulatory role for Btk in macrophages during Lm infection. In the second part of my Ph.D. thesis I initiated a comprehensive functional analysis of TFK-deficient macrophages upon activation via TLRs. For this purpose, I studied cytokine production and other effector functions of Btk-/-, Tec-/- and Btk/Tec-double-deficient BMMs in response to stimulation with various TLR ligands. The results of this part of my thesis revealed that Btk and Tec are differentially utilized in different TLR pathways and that they are further required for efficient phagocytosis of bacterial fragments. Taken together, the results of my Ph.D. thesis reveal novel roles for the TFKs Btk and Tec in different aspects of the innate immune response of macrophages to infection.submitted by Afitap Derya KöprülüAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersZsfassung in dt. SpracheWien, Med. Univ., Diss., 2013OeBB(VLID)171451

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

Tec-deficient mice are highly resistant to candidiasis.

<p>(<b>a</b>) Survival of mice after intraperitoneal infection (<i>i.p.</i>) with 5.10⁷ CfUs of <i>C. albicans</i>; for analysis of mouse survival curves Log-rank (Mantle-Cox) test was used. n = 12 per genotype. (<b>b</b>) Survival of mice after intravenous infection (<i>i.v.</i>) with 1.10⁵ CfUs of <i>C. albicans</i>; for analysis of mouse survival curves Log-rank (Mantle-Cox) test was used. n = 11 per genotype. (<b>c</b>) Histopathological analysis of mice after i.v. infection with 1.10⁵ CfUs of <i>C. albicans</i>; HE-Staining. (<b>d</b>) Intracellular staining of active caspase-8 in neutrophils (CD11b⁺Ly6C⁺Ly6G⁺), inflammatory monocytes (CD11b⁺Ly6C⁺Ly6G⁻) and inflammatory DCs (CD11b⁺CD11c⁺Ly6C⁺) in leukocytes isolated from kidneys after <i>i.v.</i> infection with 5.10⁶ CfUs of <i>C. albicans</i> for 24 h. (<b>e</b>) ELISA of respective cytokines in sera of mice after <i>i.v.</i> infection with 5.10⁶ CfUs of <i>C. albicans</i> for 24 h. Data are representative of two (<b>d,e</b>) independent experiments.</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

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

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

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

Dectin-1 is required for Tec-dependent caspase-8 activation.

<p>(<b>a</b>) Caspase-8 activity after 60 Min stimulation of BMMs with <i>C. albicans</i> (Ca), dimethylsulfoxide (DMSO), CytochalasinD (CytoD; 2 µM) or Dynasore (80 µM); chemiluminenscence of unstimulated cells, cells with respective inhibitor, cells with DMSO or <i>C. albicans</i> only was subtracted. (<b>b</b>) Caspase-8 activity after 60 Min of stimulation with <i>C. albicans</i> only (Ca) or with dimethylsulfoxide (DMSO) or BafilomycinA₁ (BafiloA₁; 30 nM) and Ca; chemiluminenscence of unstimulated cells, cells with respective inhibitor, cells with DMSO or <i>C. albicans</i> only was subtracted. (<b>c</b>) Caspase-8 activity after 60 Min of stimulation with <i>C. albicans</i> only (Ca) or with dimethylsulfoxide (DMSO), Syk Inhibitor R406 (3 µM), Src Inhibitor PP2 (5 µM) or the non-functional analogon PP3 (5 µM) and Ca; chemiluminenscence of unstimulated cells, cells with respective inhibitor, cells with DMSO or <i>C. albicans</i> only was subtracted. (<b>d</b>) Caspase-8 activity after 60 Min stimulation with <i>C. albicans</i> only (Ca) in BMMs of indicated genotype; chemiluminenscence of unstimulated cells or <i>C. albicans</i> only was subtracted. (<b>e</b>) Caspase-8 activity after 60 Min of stimulation with <i>C. albicans</i> only (Ca) in WT BMMs blocked with indicated antibodies (all 10 µg/ml) and respective isotype control (10 µg/ml); chemiluminenscence of unstimulated cells, cells treated with antibody only or <i>C. albicans</i> only was subtracted. (<b>f</b>) Immunoblot of Tec activation of cell lysates after stimulation with <i>C. albicans</i>; lysates were enriched for phospho-protein fraction using respective kit. Data are representative of at least two (<b>f</b>), three (<b>d</b>,<b>e</b>) or four (<b>a</b>–<b>c</b>) independent experiments. Mean and SD are shown.</p

Fungal β-glucans activate caspase-8 in murine BMMs.

<p>(<b>a</b>) Caspase-8 activity after 60 Min stimulation of BMMs with <i>C. albicans</i> (Ca) or curdlan (200 µg/ml); dectin-1 was blocked with laminarin (500 µg/ml); chemiluminenscence of unstimulated cells, cells with laminarin only or <i>C. albicans</i> only was subtracted. (<b>b</b>) ELISA of IL-1β in supernatants of BMMs after stimulation with <i>C. albicans</i> only (Ca) or curdlan (200 µg/ml); dectin-1 was blocked with laminarin (500 µg/ml). (<b>c</b>) Caspase-8 activity after 60 Min of stimulation with <i>C. albicans</i> only (Ca), heat-killed Ca (10 Min on 70°C)); chemiluminenscence of unstimulated cells or <i>C. albicans</i> resp. <i>Candida glabrata</i> only was subtracted. (<b>d</b>) ELISA of IL-1β in supernatants of BMMs after stimulation with <i>C. albicans</i> only (Ca), heat-killed Ca (10 Min on 70°C), <i>efg1</i>Δ/Δ or <i>efg1</i>Δ/Δ <i>cph1</i>Δ/Δ Ca-mutants or left unstimulated (-). (<b>e,f</b>) Caspase-8 activity after 60 Min of stimulation with <i>L. monocytogenes</i>. <i>S. aureus</i>, <i>P. aeruginosa</i> or <i>S. pyogenes</i>; chemiluminenscence of unstimulated cells or bacteria only was subtracted. ELISA of IL-1β in supernatants of BMMs after stimulation with <i>L. monogenes</i>. <i>S. aureus</i>, <i>P. aeruginosa</i> or <i>S. pyogenes</i> or left unstimulated (-). Data are representative of at least three independent experiments. Mean and SD are shown.</p

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