PMC6857097_01
Investigations of IL-6 functions in the patient. (A) The dynamics of IL-6, CRP, PCT, and absolute leukocytes count in various time-points during sepsis, 1 and 3 months afterwards (h-hours from onset of fever, m-month/s). An extreme elevation of PCT is not accompanied with IL-6 or CRP increases over the normal reference value. Reference ranges: CRP 0.0-5.0 mg/L; PCT 0.0-0.5 mug/L; IL-6 0.0-20.0 ng/L; Leu 6.0-17.5 x 109/L. (B) Intracellular IL-6 production in patient's monocytes at the time of sepsis compared to 59 healthy controls' monocytes after 1 mug/ml LPS determined with flow cytometry. Unstimulated state is expressed as MFI (mean fluorescence intensity). The effect of LPS stimulation is expressed as DeltaMFI (stimulated minus unstimulated MFI). The synthesis of IL-6 by patient's monocytes is unskewed. (C) Detection of extracellular IL-6 from patient's PBMCs after 1 mug/ml LPS stimulation determined by ELISA. The release of IL-6 into extracellular space by patient's cells is normal. (D) Suppression of extracellular IL-6 by patient's serum analyzed by ELISA. Healthy age-matched controls' PBMCs (n = 2) were stimulated with LPS, cultivated in complete media (CM) supplemented either with fetal bovine serum (FBS) or 10% patient serum in time of sepsis (I) and 1 month later (II) The amount of IL-6 detected in the presence of patient's serum is decreased in both time-points. (E) Anti-IL-6 autoantibodies detection in patient serum obtained in time of sepsis (I), 1 month later (II), and in 2 healthy age-matched controls with ELISA. The patient's serum, but not the control serum, contains anti-IL6 autoantibodies. OD, optical density. (F) STAT3 phosphorylation (pSTAT3) in control (n = 2) T cells and monocytes after 10 ng/ml recombinant IL-6 stimulation. The peripheral blood was stimulated with IL-6 diluted in PBS containing 20% patient serum obtained in sepsis (I), 1 month later (II), 3 months later (III), or in fetal bovine serum (FBS). The patient's serum decreases the pSTAT3 signal in all three time-points. Data are expressed as DeltaMFI (stimulated minus unstimulated MFI). MFI, mean fluorescence intensity.
fimmu-10-02629-g0001.jpg
PMC6857097_02_fimmu-10-02629-g0001.jpg
F1
We found the PBMCs of the patient to be capable of substantial IL-6 extracellular release, even if slightly decreased compared to a healthy control (Figure 1C). This indicated that the patient's serum contained a component interfering with the IL-6 detection (Figure 1D). While the control samples were negative for anti-IL6 abs, the patient's serum was found positive in time of sepsis as well as 1 month after the infection (Figure 1E). We observed a significantly lower pSTAT3 signal from samples containing patient's serum from all 3 time-points (Figure 1F).
PMC6857097_02
Investigations of IL-6 functions in the patient. (A) The dynamics of IL-6, CRP, PCT, and absolute leukocytes count in various time-points during sepsis, 1 and 3 months afterwards (h-hours from onset of fever, m-month/s). An extreme elevation of PCT is not accompanied with IL-6 or CRP increases over the normal reference value. Reference ranges: CRP 0.0-5.0 mg/L; PCT 0.0-0.5 mug/L; IL-6 0.0-20.0 ng/L; Leu 6.0-17.5 x 109/L. (B) Intracellular IL-6 production in patient's monocytes at the time of sepsis compared to 59 healthy controls' monocytes after 1 mug/ml LPS determined with flow cytometry. Unstimulated state is expressed as MFI (mean fluorescence intensity). The effect of LPS stimulation is expressed as DeltaMFI (stimulated minus unstimulated MFI). The synthesis of IL-6 by patient's monocytes is unskewed. (C) Detection of extracellular IL-6 from patient's PBMCs after 1 mug/ml LPS stimulation determined by ELISA. The release of IL-6 into extracellular space by patient's cells is normal. (D) Suppression of extracellular IL-6 by patient's serum analyzed by ELISA. Healthy age-matched controls' PBMCs (n = 2) were stimulated with LPS, cultivated in complete media (CM) supplemented either with fetal bovine serum (FBS) or 10% patient serum in time of sepsis (I) and 1 month later (II) The amount of IL-6 detected in the presence of patient's serum is decreased in both time-points. (E) Anti-IL-6 autoantibodies detection in patient serum obtained in time of sepsis (I), 1 month later (II), and in 2 healthy age-matched controls with ELISA. The patient's serum, but not the control serum, contains anti-IL6 autoantibodies. OD, optical density. (F) STAT3 phosphorylation (pSTAT3) in control (n = 2) T cells and monocytes after 10 ng/ml recombinant IL-6 stimulation. The peripheral blood was stimulated with IL-6 diluted in PBS containing 20% patient serum obtained in sepsis (I), 1 month later (II), 3 months later (III), or in fetal bovine serum (FBS). The patient's serum decreases the pSTAT3 signal in all three time-points. Data are expressed as DeltaMFI (stimulated minus unstimulated MFI). MFI, mean fluorescence intensity.
fimmu-10-02629-g0001.jpg
PMC6857097_02_fimmu-10-02629-g0001.jpg
F1
We found the PBMCs of the patient to be capable of substantial IL-6 extracellular release, even if slightly decreased compared to a healthy control (Figure 1C). This indicated that the patient's serum contained a component interfering with the IL-6 detection (Figure 1D). While the control samples were negative for anti-IL6 abs, the patient's serum was found positive in time of sepsis as well as 1 month after the infection (Figure 1E). We observed a significantly lower pSTAT3 signal from samples containing patient's serum from all 3 time-points (Figure 1F).