Serum and Serum Albumin Inhibit in vitro Formation of Neutrophil Extracellular Traps (NETs)

Abstract

The formation of neutrophil extracellular traps (NETs) is an immune defense mechanism of neutrophilic granulocytes. Moreover, it is also involved in the pathogenesis of autoimmune, inflammatory, and neoplastic diseases. For that reason, the process of NET formation (NETosis) is subject of intense ongoing research. In vitro approaches to quantify NET formation are commonly used and involve neutrophil stimulation with various activators such as phorbol 12-myristate 13-acetate (PMA), lipopolysaccharides (LPS), or calcium ionophores (CaI). However, the experimental conditions of these experiments, particularly the media and media supplements employed by different research groups, vary considerably, rendering comparisons of results difficult. Here, we present the first standardized investigation of the influence of different media supplements on NET formation in vitro. The addition of heat-inactivated (hi) fetal calf serum (FCS), 0.5% human serum albumin (HSA), or 0.5% bovine serum albumin (BSA) efficiently prevented NET formation of human neutrophils following stimulation with LPS and CaI, but not after stimulation with PMA. Thus, serum components such as HSA, BSA and hiFCS (at concentrations typically found in the literature) inhibit NET formation to different degrees, depending on the NETosis inducer used. In contrast, in murine neutrophils, NETosis was inhibited by FCS and BSA, regardless of the inducer employed. This shows that mouse and human neutrophils have different susceptibilities toward the inhibition of NETosis by albumin or serum components. Furthermore, we provide experimental evidence that albumin inhibits NETosis by scavenging activators such as LPS. We also put our results into the context of media supplements most commonly used in NET research. In experiments with human neutrophils, either FCS (0.5-10%), heat-inactivated (hiFCS, 0.1-10%) or human serum albumin (HSA, 0.05-2%) was commonly added to the medium. For murine neutrophils, serum-free medium was used in most cases for stimulation with LPS and CaI, reflecting the different sensitivities of human and murine neutrophils to media supplements. Thus, the choice of media supplements greatly determines the outcome of experiments on NET-formation, which must be taken into account in NETosis research.

Keywords: NET; NETosis; experimental conditions; in vitro experiments; media; neutrophil extracelluar traps; neutrophils.

Figure 1

Influence of serum and serum albumin supplements on NET formation of human neutrophils. (A) Representative fluorescence images of human neutrophils (chromatin stained by Hoechst) after stimulation with CaI (4 μM), PMA (100 nM), or LPS (25 μg/ml) for 180 min, respectively. NET formation of neutrophils was studied in RPMI 1,640 with 10 mM HEPES (RPMI/HEPES), RPMI/ HEPES + 0.5% human serum albumin (HSA) or + 1% heat inactivated (56°C) fetal calf serum (hiFCS). Chromatin decondensation induced by PMA is clearly visible with all three culture conditions, while LPS or CaI only cause NET formation in BSA- and HSA-free RPMI/HEPES. Scale = 50 μm. (B) Neutrophils were stimulated to undergo NET formation with CaI (4 μM), PMA (100 nM) or LPS (10, 25, or 100 μg/ml), respectively. Both, HSA and BSA inhibit CaI and LPS-induced formation of NETs (determined as percentage of decondensed nuclei/NETs of total neutrophils). NETosis stimulated by PMA is independent of serum albumin addition. Error bars = mean ± SEM. ns, not significant. *p < 0.05, ****p < 0.0001. N = 4–9 [pool = 15 donors, 6–9 (unstimulated), 4 (CaI, 25 and 100 μg/ml LPS), 4–5 (10 μg/ml LPS), 5–8 (PMA)]. Two-way-ANOVA, Bonferroni's multiple comparisons test. (C) NET formation of neutrophils stimulated in RPMI/ HEPES supplemented with 0.5, 1, or 2% heat inactivated (56°C) fetal calf serum (hiFCS). Addition of hiFCS to RPMI/ HEPES decreases the percentage of decondensed nuclei/NETs after stimulation by CaI or LPS in a dose-dependent manner. PMA-induced NET formation occurs independently of FCS addition. Error bars = mean ± SEM. ns, not significant. *p < 0.05. ****p < 0.0001. N = 3–8 [pool = 15 donors, 5–8 (unstimulated), 3–5 (100 μg/ml LPS), 4–5 (PMA, CaI, 10 and 25 μg/ml LPS)]. Two-way-ANOVA, Bonferroni's multiple comparisons test.

Figure 2

Extracellular NETotic DNA is rich in MPO. Representative fluorescence images of NETs induced by CaI (4 μM), PMA (100 nM), or LPS (25 μg/ml) activated in supplement-free RPMI/HEPES. The images show a clear colocalization of MPO (red) with the extracellular DNA-fibers (blue) of released NETs. Scale = 10 μm.

Figure 3

Influence of serum and serum albumin supplements on NET formation of murine neutrophils. (A) Representative fluorescence images of nuclei of murine neutrophils (Hoechst) after stimulation with CaI (4 μM), PMA (100 nM) or LPS (25 μg/ml) for 180 min, respectively. NET formation of neutrophils was studied in RPMI/ HEPES without supplements and RPMI/ HEPES supplemented with 0.5% BSA or 2% hiFCS, as indicated. Chromatin decondensation is only inducible in RPMI/ HEPES (white arrow heads). Scale = 50 μm. (B) Percentage of decondensed nuclei/ NETs after stimulation with PMA (100 nM), CaI (4 μM), or LPS (10, 25, or 100 μg/ml) for 180 min. Murine neutrophils were studied in RPMI/ HEPES and RPMI/ HEPES supplemented with 0.5% BSA or 2% hiFCS. Addition of 0.5% BSA or 2% hiFCS to RPMI/ HEPES, inhibits chromatin decondensation induced by PMA, CaI and LPS completely. Error bars = mean ± SEM. ns = not significant. ****p < 0.0001. N = 3–13 mice [3–13 (unstimulated), 3–5 (100 μg/ml LPS), 3–4 (CaI, 10 and 25 μg/ml LPS), 3–5 (PMA)]. two-way-ANOVA, Bonferroni's multiple comparisons test.

Figure 4

Binding of LPS to serum albumins. Box plots represent fluorescence anisotropy values for (A), bovine serum albumin (0.005%) and (B), human serum albumin (0.005%) after addition of LPS from Pseudomonas aeruginosa at 10, 25, or 50 μg/ml, as indicated. The anisotropy of albumin increases with higher LPS concentrations. For each concentration, the average anisotropy value (monitored for 500s) between two LPS additions is shown. Box plots show 25 and 75 percentiles with the midline as median, the square as arithmetic middle and crosses as 99%/1% values. Whiskers include all data points within the 1.5 interquartile range. N = 1.

Figure 5

Heterogeneity in media supplements for human in vitro NETosis studies. Pie charts of serum, plasma, or serum albumin supplements used in human in vitro NETosis studies, as indicated (numbers represent the absolute numbers of publications using a certain supplement while the slices represent percentages of all considered publications). (A) chart for all stimuli combined (all publications were counted once). (B) charts for PMA, Ionophore/ Ionomycin, LPS, pathogen and cytokines/chemokines, respectively. Publications were counted for multiple activator-specific pie charts if more than one NET-activator was used. N = 460 publications up to the 1st of March 2018.

Figure 6

Heterogeneity in media supplements for murine in vitro NETosis studies. Pie charts of serum, plasma, or serum albumin supplements used in murine in vitro NETosis studies, as indicated (numbers represent the absolute numbers of publications using a certain supplement while the slices represent percentages of all considered publication). (A) chart for all stimuli combined. (B) charts for PMA, Ionophore/ Ionomycin, LPS, pathogen, and cytokines/chemokines, respectively. Publications were counted for multiple activator-specific pie charts if more than one NET-activator was used. N = 108 publications up to the 1st of March 2018.