Hierarchical clustering of cytokine and chemokine genes by <i>M. abscessus</i>.

<p>Neutrophils were stimulated with <i>M. abscessus</i> (Sm; <i>yellow shading</i>), or <i>S. aureus</i> (Sa; <i>red shading</i>) for 2 hours, and gene expression was determined compared to non-stimulated neutrophils (C; <i>blue shading</i>). Tree spacing indicates linkage distance. Highly expressed (<i>red</i>) and low expressed (<i>green</i>) genes are indicated from 4 different neutrophil donors.</p

Killing and phagocytosis of <i>M. abscessus</i>.

<p><b>A</b>, Neutrophil killing of <i>M. abscessus</i>. Neutrophils were incubated with <i>M. abscessus</i> for 2 and 4 hours; surviving bacteria were enumerated and compared to bacterial cfu in the absence of neutrophils for the same time points to obtain percent killing. Data represents mean±SEM of 6 experiments. <b>B</b>, Phagocytosis of <i>M. abscessus</i> by neutrophils. FITC-labeled bacteria were incubated with neutrophils for the indicated times, and the percent of neutrophils with intracellular staining was determined. Data represents mean±SEM of 10 experiments. Two-way ANOVA was performed; *p<0.05 by Bonferroni post-test. <b>C</b>, Extracellular DNA release. Neutrophils were exposed to <i>M. abscessus</i> (Mab) at a MOI of 2 for 4 hours. Data represents mean±SEM; n = 13. <b>D</b>, Extracellular DNA release by neutrophils exposed to <i>S. aureus</i> (Sa) at an MOI of 2 for 2 hours. Data represents mean±SEM; n = 6. Analysis for significance for <b>C</b> and <b>D</b> was determined by a paired, one-tail t test.</p

<i>M. abscessus</i> does not alter neutrophil death pathways.

<p>Neutrophils were incubated with <i>M. abscessus</i> (Mab; <i>closed bars</i>) or <i>S. aureus</i> (Sa; <i>open bars</i>) for 4 hours. <b>A</b>, Necrotic cell death was measured by release of lactate dehydrogenase (LDH). <b>B</b>, Apoptotic cell death was measured by solubilized histone-bound DNA. Only <i>S. aureus</i> significantly changed either necrosis or apoptosis compared to non-stimulated neutrophils. Data represents mean±SEM; n = 6–9; *p<0.05, **p<0.01, †p<0.001 by Bonferroni post-test analysis.</p

<i>M. abscessus</i> biofilm density is enhanced by neutrophils.

<p>Bacteria (<i>hatched bars</i>), neutrophils (<i>open bars</i>), or the combination of bacteria and neutrophils (<i>closed bars</i>) were incubated for 5 days, and biofilm density was assessed by crystal violet staining in response to <b>A</b>, <i>M. abscessus</i>; <b>B</b>, <i>P. aeruginosa</i>. Data represent mean±SEM from 4–9 experiments. *p<0.05; †, p<0.001. Representative biofilm formation in a flow chamber by: <b>C</b>, <i>M. abscessus</i> alone (10×); <b>D</b>, <i>M. abscessus</i> and neutrophils (40×); <b>E</b>, neutrophils alone (40×). Biofilms were stained for live (<i>green</i>) and dead (<i>red</i>) cells.</p

Gene expression analysis indicates a core of commonly induced genes.

<p><b>A</b>, Total number of unique upregulated genes by <i>M. abscessus</i> (Mab), and <i>S. aureus</i> (Sa) are indicated near the organism name, and overlapping genes are indicated within sections of the Venn diagram. <b>B</b>, Downregulated genes. Analysis was performed on 4 independent experiments.</p

Secretion of chemokines and cytokines.

<p>Neutrophils were stimulated with <i>M. abscessus</i> (Mab; <i>closed bars</i>), or <i>S. aureus</i> (Sa; <i>open bars</i>) for 2 and 4 hours, or left unstimulated (NS; <i>hatched bars</i>) and supernatants were collected. The indicated cytokine and chemokine levels were determined by ELISA. Mean±SEM, n = 7–8; *p<0.05; **p<0.01; †p<0.001 by Bonferroni post-test analysis.</p

Superoxide anion release in response to <i>M. abscessus</i>.

<p><i>M. abscessus</i> (Mab; <i>closed bar</i>), or <i>S. aureus</i> (Sa; <i>open bar</i>) were added to neutrophils at a 10∶1 MOI, and superoxide release was compared to control neutrophils (<i>hatched bars</i>) by measuring reduction of cytochrome <i>c</i> after 60 min. Data is presented as mean±SEM; Bonferroni post-test indicated significant release of superoxide; n = 10, *p<0.05.</p