3.1 Summary - Manuscript I
„Chromatin Swelling Drives Neutrophil Extracellular Trap Release“
(Nature Communications, 2018)
Detailed real-time observations on single human neutrophils conducted within this study, led to the conclusion that NETosis occurs in three distinct phases (P1-P3). These phases were reproducible among different donors as well as with frequently used NET-inductors such as PMA, calcium ionophore (CaI) and LPS. The first phase (P1) depended strongly on temperature and enzyme activity as verified by time-resolved inhibition of energy generation and MPO activity. Within this phase, the cell consumed substantial amounts of ATP, started to rearrange membranes and degraded the cytoskeleton. During this process, the cell softened and decreased its membrane tension. The chromatin within the neutrophil nucleus remained condensed in P1. The second phase (P2) was characterized by chromatin decondensation and was mostly independent of enzymatic activity and temperature variation. Consequently, the main driving force of P2 was the entropic swelling of chromatin.
The onset of P2, the start of chromatin decondensation, was interestingly also accompanied by the rupture of the nuclear envelope as determined by lamin B fluorescence staining. After this time-point, NETosis could not be inhibited any more. Therefore, the onset of P2 in NETosis marks a point of no return. After complete chromatin decondensation, cytoskeleton degradation and cell rounding, the cell membrane ruptured and released the NET (P3). The point of cytoplasmic membrane rupture was biomechanically pre-determined by the position of the nucleus within the cell as well as plasma membrane dynamics during NETosis.
In summary, NETosis consists of an active first phase and a second phase driven by material properties, which are separated by a clear point of no return. These results present a detailed insight into the exact regulation of NETosis and demonstrate how material properties can drive the complex behavior of cells.
CONTRIBUTION:
General: Experimental design together with all authors; main parts of the manuscript with Sebastian Kruss, Luise Erpenbeck and Daniel Meyer (D.M.); figure arrangement; co-supervision of Susanne N.
Senger-Sander and Anja Kwaczala-Tessmann, who both contributed with selected data sets.
Data generation and analysis: Figures: Fig. 1a-e; Fig. 2a/d; Fig. 2c (imaging); Fig. 3; Fig. 4b; Fig. 4a/d (all raw data; data analysis by D.M.); Fig. 4c (sample preparation); Fig. 5a (F-actin); Fig. 5b (Latrunculin A); Fig. 5c (Jasplakinolide); Fig. 6a-e (all raw data; data analysis together with D.M.) and Fig. 7 (together with D.M.). Supplementary figures: Supp. fig. 1; Supp. fig. 2 (sample preparation);
Supp. fig. 3; Supp. fig. 4a/b; Supp. fig. 4d (together with D.M.); Supp. fig. 5; Supp. fig. 6; Supp. fig. 7;
Supp. fig. 8a; Supp. fig. 8b/c (sample preparation); Supp. fig. 9a (all raw data, data analysis by D.M.);
Supp. fig. 9c; Supp. fig. 11 (all raw data, data analysis by D.M.) and Supp. fig. 13 (together with D.M.).
Supplementary movies: movie 1-8; movie 12-15 and movie 17. Analysis by customized Matlab code
CHAPTER 3 - Summary
Dissertation - Elsa Neubert
3.2 Summary - Manuscript II
“Serum and Serum Albumin Inhibit in vitro Formation of Neutrophil Extracellular Traps (NETs)”
(Frontiers in Immunology, 2019)
As the composition of cell culture media can massively alter the results of in vitro cell studies, the effect of serum supplement in culture media on NETosis was analyzed in vitro. A complementary systematic literature search was also conducted. In vitro NETosis induced by PMA, calcium ionophore (CaI) or LPS depended strongly on supplementation of serum or serum albumin to culture media. In serum-free media, all tested stimuli activated NETosis as verified by colocalization of extracellular DNA with MPO and release of active NE-DNA-complexes. The addition of serum albumin (HSA, BSA), however, significantly inhibited CaI- and LPS-induced NETosis of human neutrophils. The same effect occurred in media supplemented with heat-inactivated fetal calf serum (hiFCS). This pronounced inhibitory effect of serum supplements could be mediated by activator binding effects, as shown for LPS and albumin by fluorescence anisotropy measurements. Remarkably, these supplements did not affect PMA-induced NETosis of human neutrophils. However, in assays with murine cells, which in general showed lower NETosis rates, serum and serum albumin were sufficient to inhibit NETosis in response to all three stimuli. This observation emphasizes the variability of NETosis in different species.
These results are of particular interest regarding the considerable variation in serum and serum albumin supplements used for in vitro NETosis studies in the literature, as shown in a comprehensive literature search. Interestingly, the choice of supplement varies according to the applied NET inducer reflecting our experimental results.
The experimental data together with the literature research corroborate the importance for the standardization of culture conditions used in in vitro NET assays in order to obtain comparable results. It also allows speculations about the remarkable susceptibility of NETosis by small variations of the neutrophil environment and therefore regulation of NETosis in vivo.
CONTRIBUTION:
General: Experimental design together with all authors; main parts of the manuscript together with Luise Erpenbeck and Susanne N. Senger-Sander (SN. S-S.); complete data plotting, statistics and figure arrangement; co-supervision of SN. S-S. (who provided the literature research and contributed to the data set of Fig. 1).
Data generation and analysis: Figures: Fig. 1 (main raw data except one selected data set by SN. S-S.); Fig. 2; Fig. 3 (main raw data, except stimulation with PMA and LPS (10 and 25 µg/ml) in RPMI/HEPES (Veit S. Manzke) and one selected data set by Sophie E.F. Scheidmann). Supplementary figures: Supp. fig. 1; Supp. fig. 2. and Supp. fig. 4. Literature research: co-supervision of SN. S-S. (who provided complete data set) and independent verification of all data.
3.3 Summary - Manuscript III
“Blue and Long-wave Ultraviolet Light Induce in vitro Neutrophil Extracellular Trap Formation”
(Prepared for submission to Frontiers in Immunology)
The effect of UV-Vis light on in vitro NET formation is the main focus of the third manuscript.
Neutrophils, freshly isolated from healthy human donors, released NETs in response to UVA and blue light. The identity of these NETs was verified by colocalization of MPO with extracellular decondensed chromatin. NE and MPO regulated this novel pathway of 'suicidal' NET formation (NETosis) supported by PAD activity. There was no involvement of necroptotic, apoptotic or RHO/ROCK-related pathways as verified by RIPK1, pan-caspase and ROCK1/2 inhibition.
Interestingly, light-induced NETosis could be activated in a locally restricted fashion and depended strongly on light doses and wavelength. While UVA and blue light (375 nm and 470 nm LEDs) revealed high NET rates, green light (565 nm LED) failed to induce NETosis.
This wavelength-dependency was reproducible for equal light energy and photon flux and correlated with the absorption spectrum of the photosensitive substance riboflavin (vitamin B2). In contrast to several previously described NETosis pathways, light-induced NETosis took place independently of mitochondrial ROS production or NADPH oxidase activation but required extracellular ROS generation. The formation of ROS was triggered by riboflavin excitation in combination with sensitizing substances such as tryptophan or HEPES buffer present in culture media.
Since riboflavin and tryptophan are present at high concentrations in human skin and UVA as well as blue light can penetrate the epidermis in significant amounts, light-induced NETosis could be relevant for skin light-sensitivity. Particularly a contribution to disease onset and exacerbation of autoimmune disorders is conceivable, especially in the context of the frequently documented dysregulation of NETosis, high ROS imbalance and increased light sensitivity in these disorders. Therefore, a more profound understanding of NETosis in response to UV-Vis light can shed light into this still enigmatic correlation.
CONTRIBUTION:
General: Experimental design together with all authors; manuscript draft; final manuscript together with Luise Erpenbeck and Katharina M. Bach (KM. B.); complete data plotting, statistics and figure arrangement; co-supervision of KM. B (who provided a main data sets for this study).
Data generation and analysis: Figures: Fig. 1; selective data for Fig. 3 (N = 5 for Cl-amidine and 4-ABAH at 375 nm); Fig. 4c; Fig. 5 (except ROS measurements in RPMIcomp. + HEPES (KM. B.)) and Fig. 6. Supplementary data: Supp. fig. 1; Supp. fig. 2a (analysis), Supp. fig 2b; Supp. fig. 3; Supp. fig. 4 and Supp. movie.