image_2_Protein Deiminase 4 and CR3 Regulate Aspergillus fumigatus and β-Glucan-Induced Neutrophil Extracellular Trap Formation, but Hyphal Killing Is Dependent Only on CR3.tiff

<p>Neutrophil extracellular trap (NET) formation requires chromatin decondensation before nuclear swelling and eventual extracellular release of DNA, which occurs together with nuclear and cytoplasmic antimicrobial proteins. A key mediator of chromatin decondensation is protein deiminase 4 (PAD4), which catalyzes histone citrullination. In the current study, we examined the role of PAD4 and NETosis following activation of neutrophils by A. fumigatus hyphal extract or cell wall β-glucan (curdlan) and found that both induced NET release by human and murine neutrophils. Also, using blocking antibodies to CR3 and Dectin-1 together with CR3-deficient CD18^−/− and Dectin-1^−/− murine neutrophils, we found that the β-glucan receptor CR3, but not Dectin-1, was required for NET formation. NETosis was also dependent on NADPH oxidase production of reactive oxygen species (ROS). Using an antibody to citrullinated histone 3 (H3Cit) as an indicator of PAD4 activity, we show that β-glucan stimulated NETosis occurs in neutrophils from C57BL/6, but not PAD4^−/− mice. Similarly, a small molecule PAD4 inhibitor (GSK484) blocked NET formation by human neutrophils. Despite these observations, the ability of PAD4^−/− neutrophils to release calprotectin and kill A. fumigatus hyphae was not significantly different from C57BL/6 neutrophils, whereas CD18^−/− neutrophils exhibited an impaired ability to perform both functions. We also detected H3Cit in A. fumigatus infected C57BL/6, but not PAD4^−/− corneas; however, we found no difference between C57BL/6 and PAD4^−/− mice in either corneal disease or hyphal killing. Taken together, these findings lead us to conclude that although PAD4 together with CR3-mediated ROS production is required for NET formation in response to A. fumigatus, PAD4-dependent NETosis is not required for A. fumigatus killing either in vitro or during infection.</p

image_1_Protein Deiminase 4 and CR3 Regulate Aspergillus fumigatus and β-Glucan-Induced Neutrophil Extracellular Trap Formation, but Hyphal Killing Is Dependent Only on CR3.tiff

<p>Neutrophil extracellular trap (NET) formation requires chromatin decondensation before nuclear swelling and eventual extracellular release of DNA, which occurs together with nuclear and cytoplasmic antimicrobial proteins. A key mediator of chromatin decondensation is protein deiminase 4 (PAD4), which catalyzes histone citrullination. In the current study, we examined the role of PAD4 and NETosis following activation of neutrophils by A. fumigatus hyphal extract or cell wall β-glucan (curdlan) and found that both induced NET release by human and murine neutrophils. Also, using blocking antibodies to CR3 and Dectin-1 together with CR3-deficient CD18^−/− and Dectin-1^−/− murine neutrophils, we found that the β-glucan receptor CR3, but not Dectin-1, was required for NET formation. NETosis was also dependent on NADPH oxidase production of reactive oxygen species (ROS). Using an antibody to citrullinated histone 3 (H3Cit) as an indicator of PAD4 activity, we show that β-glucan stimulated NETosis occurs in neutrophils from C57BL/6, but not PAD4^−/− mice. Similarly, a small molecule PAD4 inhibitor (GSK484) blocked NET formation by human neutrophils. Despite these observations, the ability of PAD4^−/− neutrophils to release calprotectin and kill A. fumigatus hyphae was not significantly different from C57BL/6 neutrophils, whereas CD18^−/− neutrophils exhibited an impaired ability to perform both functions. We also detected H3Cit in A. fumigatus infected C57BL/6, but not PAD4^−/− corneas; however, we found no difference between C57BL/6 and PAD4^−/− mice in either corneal disease or hyphal killing. Taken together, these findings lead us to conclude that although PAD4 together with CR3-mediated ROS production is required for NET formation in response to A. fumigatus, PAD4-dependent NETosis is not required for A. fumigatus killing either in vitro or during infection.</p

image_3_Protein Deiminase 4 and CR3 Regulate Aspergillus fumigatus and β-Glucan-Induced Neutrophil Extracellular Trap Formation, but Hyphal Killing Is Dependent Only on CR3.tiff

<p>Neutrophil extracellular trap (NET) formation requires chromatin decondensation before nuclear swelling and eventual extracellular release of DNA, which occurs together with nuclear and cytoplasmic antimicrobial proteins. A key mediator of chromatin decondensation is protein deiminase 4 (PAD4), which catalyzes histone citrullination. In the current study, we examined the role of PAD4 and NETosis following activation of neutrophils by A. fumigatus hyphal extract or cell wall β-glucan (curdlan) and found that both induced NET release by human and murine neutrophils. Also, using blocking antibodies to CR3 and Dectin-1 together with CR3-deficient CD18^−/− and Dectin-1^−/− murine neutrophils, we found that the β-glucan receptor CR3, but not Dectin-1, was required for NET formation. NETosis was also dependent on NADPH oxidase production of reactive oxygen species (ROS). Using an antibody to citrullinated histone 3 (H3Cit) as an indicator of PAD4 activity, we show that β-glucan stimulated NETosis occurs in neutrophils from C57BL/6, but not PAD4^−/− mice. Similarly, a small molecule PAD4 inhibitor (GSK484) blocked NET formation by human neutrophils. Despite these observations, the ability of PAD4^−/− neutrophils to release calprotectin and kill A. fumigatus hyphae was not significantly different from C57BL/6 neutrophils, whereas CD18^−/− neutrophils exhibited an impaired ability to perform both functions. We also detected H3Cit in A. fumigatus infected C57BL/6, but not PAD4^−/− corneas; however, we found no difference between C57BL/6 and PAD4^−/− mice in either corneal disease or hyphal killing. Taken together, these findings lead us to conclude that although PAD4 together with CR3-mediated ROS production is required for NET formation in response to A. fumigatus, PAD4-dependent NETosis is not required for A. fumigatus killing either in vitro or during infection.</p

MyD88 pathways in leukocytes regulate diabetes-induced generation of superoxide by the retina.

<p>Diabetes caused a doubling of superoxide production by retina in wildype mice, whereas deletion of MyD88, IL-1βr or TLR2/4 only from marrow-derived cells significantly inhibited the diabetes-induced increase in superoxide generation by the retina. n=5-8 per group.</p

Photomicrograph indicating trapped leukiocytes within the retinal vasculature (leukostasis).

<p>The number of these trapped leukocytes is summarized in Figure 2.</p

Adherence of leukocytes to the endothelia of the retinal vascular wall (leukostasis) is increased in diabetes, and occurs via MyD88-dependent signalling pathways.

<p>Wildtype leukocytes from diabetic mice attached to the vessel wall in significantly greater numbers than did leukocytes from nondiabetic wildtypes, whereas leukocytes lacking either MyD88, TLR2/4 or IL-1βr underwent less diabetes-induced leukostasis than wildtype diabetic controls. Photomicrograph is a representative picture of a diabetic wildtyype animal. n=5 per group; representative of two repeat experiments.</p

Albumin in neural retina (a possible marker of vascular permeability).

<p>The retina wsa perfused to remove blood, and albumin remaining in the retina after that perfusion was interpretd as having leaked into the neural retina. Diabetes significanlty increased that albumin accumulation in wildtpe chimeras. Deletion of IL1β or TLR2/4 or MyD88 from marrow-derived cells had no significant effect on that parameter. n=5 per group.</p

Cytokine production in diabetic retinas.

<p>Retinas from diabetic and normal mice were stimulated with TLR ligands, and retinal generation of CXCL1 and IL-6 were examined by ELISA. (A and B) retinas were incubated for 6h with 10 µg/ml of Pam3CysK (to activate TLR2), PolyI:C (TLR3), LPS (TLR4), CpG DNA (TLR9), or with IL-1ß (IL-1r). (C and D) IL-6 and CXCL1 production by retinas following incubation with media alone or containing 10 µg/ml Pam3CysK. Data are mean ± SD of three retinas per group, and are representative of two repeat experiments.</p

Intoxication of A549 lung epithelial cells by effector-negative clinical isolates of <i>P. aeruginosa</i>.

<p>Clinical isolates that do not secrete detectable levels of known effectors and translocators <i>in vitro</i> as well as defined T3SS null derivatives (Δ<i>popD</i>, Δ<i>pscD</i>, where the entire open reading frame was removed by an in-frame deletion, or <i>pscC</i>-, where the <i>pscC</i> open reading frame was disrupted by the insertion of a non-replicating plasmid) were tested for their ability to intoxicate A549 epithelial cells in a T3SS-dependent manner. Delivery of effector proteins was measured by assaying rounding of A549 cells by microscopic examination. Data presented is the mean of three independent experiments with standard deviation (error bar). A) Isolates with no significant cytotoxicity. B) Isolates that are cytotoxic in a T3SS-dependent manner. C) Isolate JT87 has no detectable T3SS-related genes by PCR. Representative phase contrast images of A549 cells infected with PAO1, PAO1 Δ<i>pscD</i> or JT87 are shown to the right of the graph in panel C. ** p<0.01, Student’s T-test.</p

Virulence of effector-negative clinical isolates of <i>P. aeruginosa</i>.

<p>Corneas of C57BL/6 mice were scarified and infected with 2*10^∧5 CFU/eye. A) Images of infected eyes were taken at 24 h and 48 h post-infection to assess corneal opacification due to infiltration of neutrophils. Corneal opacification was quantitated digitally using Metamorph software as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086829#pone.0086829-Sun1" target="_blank">[11]</a>. Opacity scores for individual eyes were plotted. The median value with interquartile range is indicated. Representative images of infected corneas are shown to the left of the graph. Statistical significance of differences was determined by Mann-Whitney test: ** p<0.01, * p<0.05 B) Mice were infected with 2*10^∧5 CFU/eye using the scratch model of corneal infection. Bacterial load (CFU/eye) was determined 48 h after infection by euthanizing the mice, removing the infected eye, homogenizing it and plating serial dilutions on BHI agar plates. Bacterial loads for individual eyes were plotted. The median value with interquartile range is indicated. ** p<0.01, * p<0.05, Mann-Whitney test.</p