Protein kinase D at the Golgi controls NLRP3 inflammasome activation

The inflammasomes are multiprotein complexes sensing tissue damage and infectious agents to initiate innate immune responses. Different inflammasomes containing distinct sensor molecules exist. The NLRP3 inflammasome is unique as it detects a variety of danger signals. It has been reported that NLRP3 is recruited to mitochondria-associated endoplasmic reticulum membranes (MAMs) and is activated by MAM-derived effectors. Here, we show that in response to inflammasome activators, MAMs localize adjacent to Golgi membranes. Diacylglycerol (DAG) at the Golgi rapidly increases, recruiting protein kinase D (PKD), a key effector of DAG. Upon PKD inactivation, self-oligomerized NLRP3 is retained at MAMs adjacent to Golgi, blocking assembly of the active inflammasome. Importantly, phosphorylation of NLRP3 by PKD at the Golgi is sufficient to release NLRP3 from MAMs, resulting in assembly of the active inflammasome. Moreover, PKD inhibition prevents inflammasome autoactivation in peripheral blood mononuclear cells from patients carrying NLRP3 mutations. Hence, Golgi-mediated PKD signaling is required and sufficient for NLRP3 inflammasome activation.PMC558412

Proteins of the Factor H protein family bind to C-reactive protein and regulate complement activity on apoptotic and necrotic host cells

The complement system protects the host against foreign organisms and induces a non-inflammatory phagocytosis of damaged or mutated host cells. Complement regulators protect intact host cells against complement attack and prevent inflammatory reactions. Factor H is the main soluble regulator of the alternative complement pathway. Factor H binds to host cells and blocks the alternative complement activation to prevent cell- and tissue damages. In addition, Factor H binds the acute phase protein C-reactive protein (CRP). CRP opsonizes apoptotic and necrotic host cells and microbial invaders for phagocytosis by activating the classical complement cascade. However, the amplification loop via the alternative pathway and the pro-inflammatory terminal complement pathway are inhibited. Here I analyzed the role of Factor H binding to CRP in this limitation of complement. Furthermore, the Factor H related (CFHR) proteins CFHR-1, CFHR-4A, and CFHR-4B, which have a high similarity to Factor H, were functionally characterized. In this work I show, that Factor H, CFHR 4A, and CFHR-4B bind different isoforms of CRP. Factor H binds monomeric CRP (mCRP), while CFHR-4A/4B bind pentameric CRP (pCRP). Moreover, the analyzed proteins of the Factor H protein family regulate complement at the surface of cellular debris. Factor H and CFHR 1 inhibit complement and protect cellular debris from inflammatory complement attack, while CFHR 4A/4B enhance the cell opsonization through recruitment of pCRP. In addition, Factor H enhances the phagocytosis of apoptotic particles and inhibits the release of the pro-inflammatory cytokine TNF-αand the chemokine IL-8. These effects of Factor H are enhanced by mCRP. Thus, Factor H, CFHR-1, CFHR-4A/4B, and CRP maintain a beneficial balance between complement activation and inhibition which is crucial to prevent inflammation and ultimately the development of autoimmune diseases

Persistence versus escape: Aspergillus terreus and Aspergillus fumigatus employ different strategies during interactions with macrophages.

Invasive bronchopulmonary aspergillosis (IBPA) is a life-threatening disease in immunocompromised patients. Although Aspergillus terreus is frequently found in the environment, A. fumigatus is by far the main cause of IBPA. However, once A. terreus establishes infection in the host, disease is as fatal as A. fumigatus infections. Thus, we hypothesized that the initial steps of disease establishment might be fundamentally different between these two species. Since alveolar macrophages represent one of the first phagocytes facing inhaled conidia, we compared the interaction of A. terreus and A. fumigatus conidia with alveolar macrophages. A. terreus conidia were phagocytosed more rapidly than A. fumigatus conidia, possibly due to higher exposure of β-1,3-glucan and galactomannan on the surface. In agreement, blocking of dectin-1 and mannose receptors significantly reduced phagocytosis of A. terreus, but had only a moderate effect on phagocytosis of A. fumigatus. Once phagocytosed, and in contrast to A. fumigatus, A. terreus did not inhibit acidification of phagolysosomes, but remained viable without signs of germination both in vitro and in immunocompetent mice. The inability of A. terreus to germinate and pierce macrophages resulted in significantly lower cytotoxicity compared to A. fumigatus. Blocking phagolysosome acidification by the v-ATPase inhibitor bafilomycin increased A. terreus germination rates and cytotoxicity. Recombinant expression of the A. nidulans wA naphthopyrone synthase, a homologue of A. fumigatus PksP, inhibited phagolysosome acidification and resulted in increased germination, macrophage damage and virulence in corticosteroid-treated mice. In summary, we show that A. terreus and A. fumigatus have evolved significantly different strategies to survive the attack of host immune cells. While A. fumigatus prevents phagocytosis and phagolysosome acidification and escapes from macrophages by germination, A. terreus is rapidly phagocytosed, but conidia show long-term persistence in macrophages even in immunocompetent hosts

Monomeric C-reactive protein modulates classic complement activation on necrotic cells

The acute-phase protein C-reactive protein (CRP) recruits C1q to the surface of damaged cells and thereby initiates complement activation. However, CRP also recruits complement inhibitors, such as C4b-binding protein (C4bp) and factor H, which both block complement progression at the level of C3 and inhibits inflammation. To define how CRP modulates the classic complement pathway, we studied the interaction of CRP with the classic pathway inhibitor C4bp. Monomeric CRP (mCRP), but not pentameric CRP (pCRP), binds C4bp and enhances degradation of C4b and C3b. Both C1q, the initiator, and C4bp, the inhibitor of the classic pathway, compete for mCRP binding, and this competition adjusts the local balance of activation and inhibition. After attachment of pCRP to the surface of necrotic rat myocytes, generation of mCRP was demonstrated over a period of 18 h. Similarly, a biological role for mCRP, C1q, and C4bp in the disease setting of acute myocardial infarction was revealed. In this inflamed tissue, mCRP, pCRP, C4bp, C1q, and C4d were detected in acetone-fixed and in unfixed tissue. Protein levels were enhanced 6 h to 5 d after infarction. Thus, mCRP bound to damaged cardiomyocytes recruits C1q to activate and also C4bp to control the classic complement pathway.-Mihlan, M., Blom, A. M., Kupreishvili, K., Lauer, N., Stelzner, K., Bergstro " m, F., Niessen, H. W. M., Zipfel, P. F. Monomeric C-reactive protein modulates classic complement activation on necrotic cells. FASEB J. 25, 4198-4210 (2011). www.fasebj.or

Liver ubiquitome uncovers nutrient-stress-mediated trafficking and secretion of complement C3

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Outcome of infection with <i>A. terreus</i> and <i>A. terreus wA</i> in immunocompromised mice.

<p>(A, B) Kaplan-Meyer survival curves analyzed by log rank test. (A) Leukopenic mice immunosuppressed with cyclophosphamide. (B) Mice immunosuppressed with cortisone acetate. (C) Histology 24 h, day 3 and day 5 post infection (p. inf.). Periodic Acid Shiff stain, fungal elements stain pink. Magnifications are indicated below the columns; scale bars represent 100 µm (10×) and 10 µm (40× and 100×). Upper row: <i>A. terreus</i> wild type. Lower row: <i>A. terreus wA</i>.</p

Changes and regulation of the C5a receptor on neutrophils during septic shock in humans

During experimental sepsis, excessive generation of the anaphylatoxin C5a results in reduction of the C5a receptor (C5aR) on neutrophils. These events have been shown to result in impaired innate immunity. However, the regulation and fate of C5aR on neutrophils during sepsis are largely unknown. In contrast to 30 healthy volunteers, 60 patients in septic shock presented evidence of complement activation with significantly increased serum levels of C3a, C5a, and C5b-9. In the septic shock group, the corresponding decrease in complement hemolytic activity distinguished survivors from nonsurvivors. Neutrophils from patients in septic shock exhibited decreased C5aR expression, which inversely correlated with serum concentrations of C-reactive protein (CRP) and clinical outcome. In vitro exposure of normal neutrophils to native pentameric CRP led to a dose- and time-dependent loss of C5aR expression on neutrophils, whereas the monomeric form of CRP, as well as various other inflammatory mediators, failed to significantly alter C5aR levels on neutrophils. A circulating form of C5aR (cC5aR) was detected in serum by immunoblotting and a flow-based capture assay, suggestive of an intact C5aR molecule. Levels of cC5aR were significantly enhanced during septic shock, with serum levels directly correlating with lethality. The data suggest that septic shock in humans is associated with extensive complement activation, CRP-dependent loss of C5aR on neutrophils, and appearance of cC5aR in serum, which correlated with a poor outcome. Therefore, cC5aR may represent a new sepsis marker to be considered in tailoring individualized immune-modulating therapy

Maturation of phagolysosomes containing <i>A. fumigatus</i> or <i>A. terreus</i> conidia.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031223#s2" target="_blank">Results</a> are shown for <i>A. terreus</i> SBUG844 and <i>A. fumigatus</i> CBS144.89 with MH-S cells. (A) FITC labeled conidia were used. Yellow signal in the merged pictures indicate co-localization of conidia with the specific phagolysosome stain. All lanes show representative fluorescence microscopy pictures. Blue: DAPI (nucleus); green: FITC labeled conidia; red: specific antibody for the phagolysosomal marker indicated on the left. Bars represent a size of 10 µm. (B) Transmission electron micrograph of phagocytosed <i>A. terreus</i> conidia 8 h and 24 h after infection. Bars represent 0.5 µm. Phagocytosed conidia are surrounded by a phospholipid bilayer at both time points.</p

Phagocytosis rates and surface exposure of galactomannan and β-1,3-glucan on resting and swollen <i>A. terreus</i> and <i>A. fumigatus</i> conidia.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031223#s2" target="_blank">Results</a> are shown for <i>A. terreus</i> SBUG844 and <i>A. fumigatus</i> CBS144.89. (A, B) Phagocytosis of conidia by alveolar macrophages (MH-S cell line). Phagocytosis rates are shown as mean+SD from three independent experiments. Statistical analysis was performed using 1-way ANOVA. * P<0.05, ** P<0.01, *** P<0.001. (A) Phagocytosis of resting and pre-swollen conidia after 0.5, 3 and 8 h of co-incubation. (B) Phagocytosis of pre-swollen conidia over 12 h. (C) FACS analysis of galactomannan and (D) β-1,3-glucan exposure. Histograms (one representative result from three independent experiments is shown) and scatter plots of median fluorescence intensity are shown (mean ± SD, three independent experiments). Statistical analysis was performed using paired t-test. * P<0.05; ** P<0.01; *** P<0.001. Dashed and dotted lines in the histogram show the respective isotype controls while the full lines represent specific fluorescence for <i>A. fumigatus</i> (thin) and <i>A. terreus</i> (thick).</p

<i>A. fumigatus</i> and <i>A. terreus</i> survival, host cell damage and hyphal growth in co-incubation with macrophages.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031223#s2" target="_blank">Results</a> are shown for <i>A. terreus</i> SBUG844 and <i>A. fumigatus</i> CBS144.89. (A–C) Survival of <i>A. fumigatus</i> and <i>A. terreus</i> upon co-incubation with macrophages. Data is shown as mean+SD from three independent experiments; statistical analysis by 1-way ANOVA and Tukey's multiple comparison test. * P<0.05; ** P<0.01; *** P<0.001. (A, B) Co-incubation with MH-S cells and (C) with bone marrow-derived murine macrophages (A) Survival of <i>A. terreus</i> and <i>A. fumigatus</i> determined by fluorescence microscopy using the metabolic dye FUN1. (B, C) Survival determined by CFU. (D, E) Relative cytotoxicity of <i>A. terreus</i> and <i>A. fumigatus</i> conidia to (D) MH-S cells and (E) human monocyte-derived macrophages determined by release of lactate dehydrogenase (LDH). Mean values+SD from three independent experiments; statistical analysis was performed per time point by unpaired, two-tailed t-test. * P<0.05; ** P<0.01; *** P<0.001 (F) Microscopic picture showing germination and filamentous growth of <i>A. fumigatus</i> and <i>A. terreus</i> 12 h (left) and 24 h (right) after infection. Scale bars = 40 µm.</p