CARD9 is critical for the generation of neutrophil-targeted chemokines in the infected CSF but it is dispensable for intrinsic chemotaxis of human neutrophils.

<p><b>(A)</b> Pooled data showing chemotaxis of healthy donor (n = 40) and patient (n = 2) neutrophils towards IL-8 (left panel) and fMLP (right panel) assessed by the EZ-TAXIScan system. <b>(B)</b> EZ-TAXIScan data for individual neutrophils moving towards either IL-8 (left panels) or fMLP (right panels); individual tracks are represented by different colours and were recorded over 1 hour. <b>(C)</b> Chemotaxis of healthy donor (n = 13–15) and patient (n = 2) neutrophils towards fMLP, C5a, IL-8 and LTB4 at the indicated concentrations, assessed by the 96-well Neuroprobe chamber. Data is analyzed by two-way ANOVA (with Bonferroni correction). <b>(D)</b> Levels of neutrophil-targeted chemokines in the CARD9-deficient patient’s infected CSF (n = 3). The range of IL-8 in the CSF found in our CARD9^+/+ patient with <i>Candida</i> meningitis post-Ommaya reservoir placement (grey bar) and previously reported for neutrophilic meningitis in patients without CARD9 deficiency (red bar) are also shown. <b>(E)</b> The patient’s infected CSF is not chemotactic toward healthy donor and patient neutrophils, as assessed by the 96-well Neuroprobe chamber (n = 2). Resting indicates neutrophil migration toward buffer; nd: not detected. <b>(F)</b> The CSF of the CARD9^+/+<i>Candida</i>-infected patient post-Ommaya reservoir, but not the CSF of the CARD9-deficient patient, is chemotactic toward healthy donor neutrophils, as assessed by the 96-well Neuroprobe chamber (n = 2–4). Resting indicates neutrophil migration toward buffer. **<i>P</i> < 0.01 by unpaired t-tests. All quantitative data represent mean ± SEM. fMLP, formyl-methionyl-leucyl-phenylalanine; LTB4, leukotriene B4; PMNs, neutrophils.