PMC5303735_01
Functional response capacity of PMNs from controls and patient. (A,B) Expression of CD62L and CD11B on PMNs surface was measured after incubation of whole-blood samples at 37 C for 45 min with PBS. One histogram representative of CD62L (A) and CD11b (B) expression at the surface of PMNs from control (white) and patient Dec 2013 (black). Expression of CD62L (C) and CD11b (D) on PMN surfaces was measured after incubation of whole-blood samples at 37 C for 45 min with PBS, Pam3CSK4 (TLR1/2 agonist, 1 mug/ml), LPS (TLR4 agonist, 10 ng/ml), TNF (5 ng/ml), GG (20 mug/ml), or germling conidia from A. fumigatus (2 x 105/ml). Samples were then stained with PE-anti-CD11b and APC-anti-L-selectin Abs at 4 C for 30 min. Results are expressed as mean fluorescence intensity (MFI). All measurements were performed in controls (n = 10, values are means +- sem) and in the patient at several different times. Each experiment in the patient was matched, at the same time, with an experiment in a healthy control with results in the normal range. Statistical significance determined by the nonparametric Mann-Whitney test is indicated. ***Significantly different from sample incubated with PBS p < 0.001.
fmicb-08-00203-g0001.jpg
PMC5303735_01_fmicb-08-00203-g0001.jpg
F1
A. fumigatus germling conidia (Figure 1C), CD11b increased moderately or not at all (depending on the stimulant), compared with unstimulated PMNs (Figures 1B,D). Also as expected (Campillo-Gimenez et al.,; Robinet et al.,), pre-treatment of whole blood from controls with TLR2, TLR4, or TNFalpha, or with GG or A. fumigatus germling conidia, followed by stimulation with fMLP, a structural analog of bacterial metabolic products, strongly increased the production of reactive oxidative species (ROS) by PMNs. We have observed the progressive restoration of his CD4 count and homeostasis of the CD8+ T cells and PMN defects (Table 1, Figure 1).
ROS production and apoptosis of PMNs from controls and patient. (A,B) ROS production by PMNs was studied by DHE oxidation; (A) ROS production by PMNs was measured after pretreatment of whole-blood samples for 45 min with TLR4 agonist and incubation for 5 min with fMLP (10-6 M). One histogram representative of ROS production by stimulated PMNs from control (white) and patient (Dec 2013) (black). (B) ROS production was measured after pretreatment of whole-blood samples for 45 min with PBS, Pam3CSK4 (TLR1/2 agonist, 1 mug/ml), LPS (TLR4 agonist, 10 ng/ml), TNF (5 ng/ml), GG (20 mug/ml), or germling conidia from A. fumigatus, and followed by fMLP (10-6 M, 5 min). Results are expressed as MFI. (C,D) Apoptosis was quantified by staining with annexin V and 7-aminoactinomycin-D. (C) PMN apoptosis was analyzed by incubating whole-blood samples in 24-well tissue culture plates at 37 C with 5% CO2 with TLR4 agonist. One cytogram representative of PMN death in control and in patient (Dec 2013). (D) PMN death was analyzed immediately after sampling (PMN spontaneous apoptosis, T0h) and by incubating whole-blood samples in 24-well tissue culture plates at 37 C with 5% CO2 with PBS, Pam3CSK4 (TLR1/2 agonist, 1 mug/ml), LPS (TLR4 agonist, 10 ng/ml), or germling conidia from A. fumigatus for 20 h at 37 C (PMN survival, T20h). PMNs were identified as CD15high cells and 2 x 105 events were counted per sample. Results are expressed as percentages of apoptotic cells. All measurements were performed in controls (n = 10, values are means +- sem) and in the patient at several different times. Each experiment in the patient was matched, at the same time, with an experiment in a healthy control with results in the normal range. Statistical significance determined by the nonparametric Mann-Whitney test is indicated. ***Significantly different from sample incubated with PBS p < 0.001.
fmicb-08-00203-g0002.jpg
PMC5303735_01_fmicb-08-00203-g0002.jpg
F2
Incubation of whole blood with the different agonists had little stimulatory effect on ROS production by the patient's PMNs (Figures 2A,B). Finally, immediately after sampling (T0h), the percentage of circulating apoptotic PMNs from controls was <2%, while for the patient it reached 32% (Figure 2D). Such PMN survival was not observed in the patient (Figures 2C,D).