Function of tec family kinases in the innate immmune response to fungal pathogens

Der human-pathogene Pilz Candida albicans (Ca) ist momentan der vierthäufigste Kankenhauskeim. Systemische Infektionen mit Candida albicans zeigen eine Mortalitätsrate von etwa 40%. Unterschiedliche Risikofaktoren tragen zur Infekton bei: sowohl basale Krankheiten (etwa HIV) als auch ein geschwächtes Immunsystem. Eine wichtige Gruppe intrazellulärer Signal-Kinasen stellen die nicht rezeptor-assoziierten Tyrosinkinasen der Tec Familie (Bmx, Btk, Itk, Rlk und Tec) dar. Mitglieder diese Familie werden hauptsächlich in Zellen des Hämatopoetischen-Systems exprimiert, somit auch in Zellen des angeborenen Immunsystems (Monozyten/Makrophagen, Dendritische Zellen, etc.). Der Verlust von oder Mutationen in Kinasen der Tec Familie verursachen unterschiedlichste Immundeffekte bei Mensch und Maus, unter anderem Störungen in B-Zell und T-Zell Funktion. Interessanterweise ist über die genaue Rolle dieser Kinasen in Zellen des angeborenen Immunsystems sehr wenig bekannt und über eine etwaige Rolle der Kinasen während Pilzinfektionen ist bis zum heutigen Tag nichts berichtet worden. Diese Arbeit berichtet zum ersten Mal über eine zelltyp-spezifische Rolle der Tec Kinasen in Infektionen mit Ca. Tec Kinasen-defiziente primäre Makrophagen aus dem Knochenmark zeigen bei Infektionen mit Candida spp. eine reduzierte Produktion von reaktiven Sauerstoff Molekülen (ROS), reduzierte Produktion von inflammatorischen Zytokinen und eine erhöhte Überlebensrate von Ca. Im Gegensatz dazu zeigen Tec Kinasen-defiziente primäre Dendritische Zellen ein verändertes Zytokin-Profil, welches große Bedeutung bei der Polarisierung von naiven T Zellen haben könnte und damit möglicherweise den Infektionsverlauf beeinflusst. Erste in vivo Versuche mit Tec Kinasen-defizienten Mäusen zeigten außerdem eine deutlich verbesserte Überlebensrate von Tec -/- Mäusen, veränderte Keimzahlen an Ca und eine mögliche Induktion einer starken tolerogenen Immunantwort.The human fungal pathogen Candida albicans (Ca) represents the 4th-most frequent cause of nosocomial bloodstream infections causing mortality rates of about 40%. Different risk factors contribute and facilitate infections with Ca: basal diseases (like HIV), as well as impaired immunity. One group of intracelullar signaling molecules involved in this process is the Tec kinase family of non-receptor tyrosine kinases (Bmx, Btk, Itk, Rlk and Tec), which are preferentially expressed in cells of the haematopoietic system, including innate immune cells (monocytes/macrophages, dendritic cells e.g.). Deficiencies in Tec family kinases cause several immunological defects in humans and mice, including defects in B cell and T cell function. However, very little is known about the role and function of Tec family kinases in innate immune cells and nothing is known about their possible role in fungal-host interactions. Here, we show that mouse innate immune cells of Tec kinase-deficient mice show cell type-specific changes in the immune response to Candida. Primary bone marrow-derived macrophages from Tec kinase knock-out mice exhibit reduced Ca killing capacity, diminished production of inflammatory cytokines and reactive oxygen species (ROS) upon infection with Candida. In contrast, bone marrow-derived myeloid dendritic cells show changes in the cytokine response, possibly altering T cell polarization which in turn influences the outcome for the host. Moreover, in vivo experiments in a mouse model of disseminated candidiasis revealed improved survival of Tec-/- mice. Fungal burdens in kidneys are altered and implicate a preferential induction of a tolerogenic immune response to Candida albicans

Intracellular kinases and ubiquitin in anti-fungal immunity

submitted by Florian ZwolanekZusammenfassung in deutscher SpracheAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersMedizinische Universität Wien, Dissertation, 2016OeBB(VLID)278978

Type I Interferons Promote Fatal Immunopathology by Regulating Inflammatory Monocytes and Neutrophils during <em>Candida</em> Infections

<div><p>Invasive fungal infections by <em>Candida albicans</em> (Ca) are a frequent cause of lethal sepsis in intensive care unit patients. While a contribution of type I interferons (IFNs-I) in fungal sepsis remains unknown, these immunostimulatory cytokines mediate the lethal effects of endotoxemia and bacterial sepsis. Using a mouse model lacking a functional IFN-I receptor (<em>Ifnar1^−/−</em>), we demonstrate a remarkable protection against invasive Ca infections. We discover a mechanism whereby IFN-I signaling controls the recruitment of inflammatory myeloid cells, including Ly6C^hi monocytes and neutrophils, to infected kidneys by driving expression of the chemokines CCL2 and KC. Within kidneys, monocytes differentiate into inflammatory DCs but fail to functionally mature in <em>Ifnar1^−/−</em> mice, as demonstrated by the impaired upregulation of the key activation markers PDCA1 and iNOS. The increased activity of inflammatory monocytes and neutrophils results in hyper-inflammation and lethal kidney pathology. Pharmacological diminution of monocytes and neutrophils by treating mice with pioglitazone, a synthetic agonist of the nuclear receptor peroxisome proliferator-activated receptor-γ (PPAR-γ), strongly reduces renal immunopathology during Ca infection and improves mouse survival. Taken together, our data connect for the first time the sepsis-promoting functions of IFNs-I to the CCL2-mediated recruitment and the activation of inflammatory monocytes/DCs with high host-destructing potency. Moreover, our data demonstrate a therapeutic relevance of PPAR-γ agonists for microbial infectious diseases where inflammatory myeloid cells may contribute to fatal tissue damage.</p> </div

IFNs-I regulate detrimental Ly6C^hi monocyte and neutrophil activity.

<p>Model of IFN-I-mediated monocyte/neutrophil recruitment and activation of inflammatory DC during invasive Ca infections. Ca recognition triggers an IFN response, which controls the production of various chemokines, including CCL2 and KC, at different anatomical body sites (BM and kidneys). In response to local CCL2 in the BM, Ly6C^hi monocytes exit into the blood stream and migrate towards the target organ where they differentiate into inflammatory DCs. To fully functionally mature and become iNOS-producing cells, DCs require signaling through IFNAR1. The high presence and activity of inflammatory DCs and neutrophils in the kidney during the early infection phase promotes a secondary strong influx of neutrophils culminating in lethal immunopathology. The suppressive action of pioglitazone on Ly6C^hi monocyte/neutrophil recruitment and function ameliorates hyper-inflammation and kidney pathology.</p

The histone chaperone HIR maintains chromatin states to control nitrogen assimilation and fungal virulence

Adaptation to changing environments and immune evasion is pivotal for fitness of pathogens. Yet, the underlying mechanisms remain largely unknown. Adaptation is governed by dynamic transcriptional re-programming, which is tightly connected to chromatin architecture. Here, we report a pivotal role for the HIR histone chaperone complex in modulating virulence of the human fungal pathogen Candida albicans. Genetic ablation of HIR function alters chromatin accessibility linked to aberrant transcriptional responses to protein as nitrogen source. This accelerates metabolic adaptation and increases the release of extracellular proteases, which enables scavenging of alternative nitrogen sources. Furthermore, HIR controls fungal virulence, as HIR1 deletion leads to differential recognition by immune cells and hypervirulence in a mouse model of systemic infection. This work provides mechanistic insights into chromatin-coupled regulatory mechanisms that fine-tune pathogen gene expression and virulence. Furthermore, the data point toward the requirement of refined screening approaches to exploit chromatin modifications as antifungal strategies

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 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

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

IFN-I signaling regulates Ly6C^hi monocytes recruitment and activation.

<p>(A) Expression of Ly6C and CD11c on GM-CSF-differentiated BM-DCs at day 8 of culture (dot plot). BM-DCs of the indicated genotypes were stimulated with heat-inactivated Ca. After 24 h, CCL2, CCL7, KC, and MIP-2 release was determined by ELISA or a multiplex bead array system. Data presented show the mean ± SEM of three independent experiments. (B–H) Mice of the indicated genotype were injected with a lethal dose of 1×10⁵ cfus Ca. At indicated time points, blood, BM and kidneys were collected. (B) Ca cfus in BM were determined and expressed as cfus/mouse bones (n = 3–5 mice per group). Each symbol represents one mouse; horizontal bars indicate the calculated median. (C) BM from day 1-infected mice was isolated and placed in culture. At indicated time points, CCL2 release into the media was measured (n = 3 mice per group). (D) Expression of Ly6C and CD11b on blood leukocytes at day 5 of Ca infection in WT versus <i>Ifnar1^−/−</i> mice. Inflammatory monocytes were gated in R1. (E) Quantification of cells in the R1 gate at different time points post infection. Depicted are percent inflammatory monocytes of total WBCs (left) and absolute cell numbers in ×10⁷/l blood (right). (n = 3–5 mice per group) (F–G) Kidney leukocytes were enriched and characterized by multi-label flow cytometry. (F) Expression of Ly6C and CD11b on kidney leukocytes at day 1 of Ca infection. Inflammatory monocytes are gated in R2 (dot plot). Expression of the inflammatory DC surface markers CCR2, CD11c, MHC II, and PDCA1 by cells in the R2 gate. Solid lines, staining of R2 cells of day 1-infected mice; shaded histograms, for CCR2: staining with isotype control antibody, for CD11c and MHCII: staining of CD11c⁻MHCII⁻ neutrophils, for PDCA1: staining of R2 cells of uninfected mice. Data presented are representatives of three independent experimental repeats (total n = 11–14 mice per group). (G) Kidney leukocytes at day one of infection were stained intracellularly for iNOS. Graphs show iNOS⁺ cells (green) overlayed on total CD45⁺ leukocytes (black). Bar diagram shows the quantification of iNOS⁺ cells in percent of total CD11b⁺ cells. Data presented show the mean ± SEM of two independent experiments (n = 8–9 mice per group). (H) Kidney total RNA was analysed for gene expression of iNOS. Data presented show the mean ± SEM of three independent experiments (n = 7–12 mice per group).</p

Pioglitazone suppresses lethal inflammatory phagocyte activity.

<p>WT mice were injected with a lethal dose of 1×10⁵ cfus Ca and treated daily with 5 mg/kg pioglitazone. (A) Survival and mean percentage of original body weight of drug-treated versus vehicle-treated mice are presented. Data presented show the sum of two independent experimental repeats (n = 13–16 mice per group). (B) Kidney leukocytes were enriched and absolute numbers of inflammatory monocytes and neutrophils determined. Each symbol represents one mouse; horizontal bars indicate the calculated median (n = 6 mice per group). (C) Kidney leukocytes at day 1 of infection were intracellularly stained for iNOS. Bar diagram shows the quantification of iNOS⁺ cells in percent of total CD11b⁺ cells, mean ± SEM (n = 6 mice per group). (D) Absolute numbers of neutrophils in kidneys of treated vs non-treated mice. Each symbol represents one mouse; horizontal bars indicate the calculated median (n = 4–5 mice per group). (E) Urea concentration in serum, mean ± SEM (n = 4–5 mice per group). (F) BM-DCs were pre-treated overnight with indicated concentrations of pioglitazone and stimulated with heat-inactivated Ca the next day. After 24 h of Ca co-incubation, CCL2, CCL7, and IFN-β release were measured by ELISA. Data presented show the mean ± SEM of 3 independent experiments.</p