Caspase cleavage of the amyloid precursor protein is prevented after overexpression of bcl-2 in a triple transgenic mouse model of Alzheimer's disease

A recent study demonstrated the lack of beta-amyloid (Aβ) plaque formation and accumulation of the amyloid precursor protein (APP) in a triple transgenic mouse model of Alzheimer's disease (3xTg-AD) following overexpression of the anti-apoptotic protein, Bcl-2 (Rohn et al., J. Neurosci. 28: 3051–9, 2008). The supposition from that study was the accumulation of APP resulted from a decrease in caspase-mediated processing of APP. To determine a direct role for the caspase-cleavage of APP in 3xTg-AD mice, we designed a site-directed caspasecleavage antibody to APP and demonstrated it is a specific marker for caspase-cleaved APP. Application of this antibody revealed neuronal staining in the hippocampus and subiculum of 3xTg-AD mice. These results were confirmed utilizing a similar site-directed antibody to caspase-cleaved APP (APPneo). The caspase cleavage of APP as well as the formation of extracellular Aβ plaques was prevented in 3xTg-AD animals overexpressing Bcl-2. These results provide further support that caspases play a proximal role in promoting the pathology associated with AD

Tracing Conidial Fate and Measuring Host Cell Antifungal Activity Using a Reporter of Microbial Viability in the Lung

Fluorescence can be harnessed to monitor microbial fate and to investigate functional outcomes of individual microbial cell-host cell encounters at portals of entry in native tissue environments. We illustrate this concept by introducing fluorescent Aspergillus reporter (FLARE) conidia that simultaneously report phagocytic uptake and fungal viability during cellular interactions with the murine respiratory innate immune system. Our studies using FLARE conidia reveal stepwise and cell-type-specific requirements for CARD9 and Syk, transducers of C-type lectin receptor and integrin signals, in neutrophil recruitment, conidial uptake, and conidial killing in the lung. By achieving single-event resolution in defined leukocyte populations, the FLARE method enables host cell profiling on the basis of pathogen uptake and killing and may be extended to other pathogens in diverse model host organisms to query molecular, cellular, and pharmacologic mechanisms that shape host-microbe interactions

Compartment-specific and sequential role of MyD88 and CARD9 in chemokine induction and innate defense during respiratory fungal infection.

Aspergillus fumigatus forms ubiquitous airborne conidia that humans inhale on a daily basis. Although respiratory fungal infection activates the adaptor proteins CARD9 and MyD88 via C-type lectin, Toll-like, and interleukin-1 family receptor signals, defining the temporal and spatial pattern of MyD88- and CARD9-coupled signals in immune activation and fungal clearance has been difficult to achieve. Herein, we demonstrate that MyD88 and CARD9 act in two discrete phases and in two cellular compartments to direct chemokine- and neutrophil-dependent host defense. The first phase depends on MyD88 signaling because genetic deletion of MyD88 leads to delayed induction of the neutrophil chemokines CXCL1 and CXCL5, delayed neutrophil lung trafficking, and fatal pulmonary damage at the onset of respiratory fungal infection. MyD88 expression in lung epithelial cells restores rapid chemokine induction and neutrophil recruitment via interleukin-1 receptor signaling. Exogenous CXCL1 administration reverses murine mortality in MyD88-deficient mice. The second phase depends predominately on CARD9 signaling because genetic deletion of CARD9 in radiosensitive hematopoietic cells interrupts CXCL1 and CXCL2 production and lung neutrophil recruitment beyond the initial MyD88-dependent phase. Using a CXCL2 reporter mouse, we show that lung-infiltrating neutrophils represent the major cellular source of CXCL2 during CARD9-dependent recruitment. Although neutrophil-intrinsic MyD88 and CARD9 function are dispensable for neutrophil conidial uptake and killing in the lung, global deletion of both adaptor proteins triggers rapidly progressive invasive disease when mice are challenged with an inoculum that is sub-lethal for single adapter protein knockout mice. Our findings demonstrate that distinct signal transduction pathways in the respiratory epithelium and hematopoietic compartment partially overlap to ensure optimal chemokine induction, neutrophil recruitment, and fungal clearance within the respiratory tract

MyD88 is required for the first phase of CXCL1 induction in the lung.

<p>WT and MyD88^(−/−) mice were challenged with 3 × 10⁷ conidia and lung tissues were processed for (A) <i>in situ</i> mRNA hybridization or (B, C) chemokine analysis 10 h p.i. (A) The panels show representative WT (top row) and MyD88^(−/−) (bottom row) lung sections hybridized with ³⁵S-labeled, CXCL1- (left column) and CXCL2-specific (right column) riboprobes and counterstained with hematoxylin. Representative micrographs from an experiment with 3 mice per group are shown at original magnification, 200×. (B) Lung or (C) BALF cytokines 10 h p.i. expressed as the fold change (+SEM) in the MyD88^(−/−) response compared to the WT response pooled from 2–3 experiments with 6–12 mice per genotype.</p

MyD88 is critical for survival, fungal clearance, and lung integrity during <i>A. fumigatus</i> challenge.

<p>WT and MyD88^(−/−) mice were challenged with 7 × 10⁷ conidia and (A) monitored for survival (Kaplan-Meier survival plot of WT (black circles; n = 9) and MyD88^(−/−) (grey circles; n = 9) mice), assayed for (B) lung fungal burden, (C) BALF albumin, and (D) BALF LDH levels at 48 h p.i. (A) One of three experiments shown. (B-D) The bar graphs show mean (+SEM) values from an experiment with 6–7 mice per genotype. (C) nd = none detected. The value was below the limit of detection of the albumin assay (dashed line).</p

Interleukin-1 receptor signaling controls MyD88-dependent chemokine induction and neutrophil recruitment.

<p>Mean (+SEM) of (A, C) BALF, (B, D) lung neutrophil recruitment and (E, G, I) BALF and (F, H, J) lung, (E, F) CXCL1, (G, H) CXCL2 and (I, J) CXCL5 levels in WT (black bars), IL-1R^(−/−) (bars with diagonal stripes), IL-18R^(−/−) (bars with crosshatch), TLR2^(−/−) (dark grey bars), and TLR4^(−/−) (light grey bars) mice 10 h p.i. with 3 × 10⁷ conidia. Data are from 2 (A-D) or 1 (E-J) experiment(s) with 5–7 mice per genotype in each experiment. Graphs from a single experiment (out of three independent experiments) are shown for E, G and I.</p

MyD88 mediates the first stage of neutrophil recruitment to the lung.

<p>(A-B) The bar graphs show the mean (+SEM) (A) lung and (B) BALF neutrophil numbers 10 h and 36 h p.i. in C57BL/6 (black bars) and MyD88^(−/−) (grey bars) mice challenged with 3 × 10⁷ conidia. Data were pooled from 3 independent experiments and include 11–14 mice per group per time point. (C) The flowplots show the frequencies of CD45.1⁺ MyD88^(+/+) and CD45.2⁺ MyD88^(−/−) neutrophils in the BM, blood, lung and BALF in a representative bone marrow chimeric mouse 10 h p.i. with 3 × 10⁷ conidia. CD45.1⁺CD45.2⁺ recipient mice were lethally irradiated and reconstituted with a 3:1 ratio of CD45.2⁺ MyD88^(−/−) to CD45.1⁺ MyD88^(+/+) BM cells. The plots were gated on CD11b⁺Ly6G⁺ neutrophils. (D) The graph shows the mean ratio (±SEM) of MyD88^(−/−) to WT neutrophils (NFs) in BM, blood, lung and BALF pooled from two experiments with 7 mice.</p

CARD9-dependent induction of ELR⁺ chemokines <i>in vitro</i> and <i>in vivo</i>.

<p>(A) The plots show mean (+SEM) CXCL1 and CXCL2 secretion by WT (black bars) or CARD9^(−/−) (light grey bars) BMMs following stimulation with <i>A. fumigatus</i> germlings (MOI = 1) as measured by ELISA. Data are from 4–5 replicates per condition from a representative experiment. (B) Strategy to generate CXCL2 reporter mouse. The graph shows CXCL2 (black lines) and mean GFP fluorescence (green lines) in transgene-positive (circle) and transgene-negative (non-Tg, square) littermates that were administered indicated amounts of Pam₃Cys₄ i.p. (C) The plots show neutrophils (left panel), inflammatory monocytes (middle panel) and CD11b⁺ DCs (right panel) that were isolated from CXCL2-GFP transgenic mice (upper panel) and non-transgenic littermates (lower panel) and analyzed for GFP expression. Representative data from 2 experiments is shown. Mice were administered 3 × 10⁷ conidia and lung cell suspensions were analyzed 36 h p.i. (D) The graphs show mean number (+SEM) of GFP⁺ lung neutrophils, inflammatory monocytes, or CD11b⁺ DCs from Tg⁺ CARD9^(+/+) (black bars), Tg⁺ CARD9^(−/−) (grey bars), Tg⁻ CARD9^(+/+) (black crosshatched bars) and Tg⁻ CARD9^(−/−) mice (grey crosshatched bars).</p

Effects of MyD88 and CARD9 are additive in murine defense against aspergillosis.

<p>(A-B) WT (black bars), MyD88^(−/−) (dark grey bars), CARD9^(−/−) (light grey bars), and MyD88^(−/−)CARD9^(−/−) (white bars) mice were challenged with 3 × 10⁷ conidia and analyzed for neutrophil recruitment in (A) lung and (B) BALF at indicated time points. Data are pooled from 3 experiments with 10–15 mice per group and are expressed as the fold change when compared to the WT control group. To compare data on neutrophil recruitment from all four genotypes from multiple experiments, we pooled the experiments performed using WT, MyD88^(−/−) and MyD88^(−/−)CARD9^(−/−) double knockout with our previously published results from WT, CARD9^(−/−) and Dectin-1^(−/−) experiments [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004589#ppat.1004589.ref023" target="_blank">23</a>]. (C) Kaplan-Meier survival plot of WT (n = 9), MyD88^(−/−) (n = 8), CARD9^(−/−) (n = 6) and MyD88^(−/−)CARD9^(−/−) (n = 9) mice infected with 3 × 10⁷ conidia. (D) Representative micrographs of H&E and GAS stained lung sections from MyD88^(−/−)CARD9^(−/−) mice 48 h p.i. Images were captured at (Di) 20× and (Dii-Diii) 40× magnification. Arrow indicates region with alveolar edema and fungal hyphae. Br = bronchiole, V = vessel.</p

CXCL1 is controlled by MyD88 in lung epithelial cells and prolongs survival in MyD88^(−/−) mice following <i>A. fumigatus</i> challenge.

<p>(A) BALF (B) lung neutrophil recruitment in WT → WT (black bars), MyD88^(−/−) → WT (dark grey bars), WT → MyD88^(−/−) (light grey bars), and MyD88^(−/−) → MyD88^(−/−) (white bars) BM chimeric mice 10 h p.i. with 3 × 10⁷ conidia. Data are expressed as the fold change when compared to the WT → WT group and were pooled from 3 experiments with 12–15 mice per group. (C) BALF and (D) lung neutrophil recruitment in IL1R^(−/−) → WT (black circles), and WT → IL1R^(−/−) (white circles) BM chimeric mice 10 h p.i. with 3 × 10⁷ conidia. Data are expressed as mean (±SEM) and are from an experiment with 9 mice per group. (E-H) Mean (+SEM) BALF (E) neutrophil recruitment, (F) CXCL1, (G) CXCL2 and (H) CXCL5 levels, in MyD88^{(−/−) CC10-MyD88} (CC10-MyD88⁺; black bars) and in MyD88^(−/−) transgene-negative littermate controls (CC10-MyD88⁻; grey bars) 10 h p.i. with 3 × 10⁷ conidia. Data were pooled from 2 experiments and include 7–9 mice per genotype. (I) Kaplan-Meier survival plot of MyD88^(−/−) mice challenged with 6–7 × 10⁷ conidia and treated 4 h p.i. with 50 ng rCXCL1 (white circles, n = 11), or PBS vehicle (grey circles, n = 12). Data were pooled from 2 experiments (p = 0.026, Gehan-Breslow-Wilcoxon test).</p