Active modulation of NETosis dynamics

4.1.1 Variations in NETosis activation

As discussed in manuscript I, NETosis has three distinct phases, of which only the first phase (P1) depends on active enzyme-driven processes. NETosis also relies immensely on external factors. Whether a specific activator induces NETosis can depend on access of the stimulus to its target, pre-activation of the cell or requirement for co-stimulation. For instance, as shown in manuscript II, even a small amount of serum or serum albumin within culture media can effectively inhibit NET formation of human and mouse neutrophils (manuscript II, Fig. 1 and 3). It is reasonable to assume that these interactions are mainly due to the binding of the activator to serum proteins [361, 362] as discussed in manuscript II and verified for the binding of LPS to serum albumin (manuscript II, Fig. 4). Besides direct activator binding, other mechanisms are also conceivable to explain the obtained results. The serum proteins studied in manuscript II can attach to the cell surface or assay substrate and thereby restrict the access of the stimulus to its receptors or intervene with cell adhesion. Indeed, serum, as well as serum albumin, can strongly decrease the spreading area of cells as shown for neutrophils on human serum albumin (HSA)-coated surfaces [363] or hamster kidney cells in

used NET stimulus known for its inconsistent activation in in vitro NET assays. NETosis induced by LPS depends on Mac-1 activation as demonstrated by inhibition of the integrin subunit M (CD11b) [281]. In a current study, we were able to corroborate these findings.

Induction of NETosis with LPS directly correlated with the spreading area of neutrophils on their substrate (Erpenbeck, Gruhn et al., preprint uploaded to bioRxiv [365], data not shown). These experiments were performed by using different substrate stiffnesses (indicated by the Young’s (E) modulus) together with collagen I- or fibronectin coatings.

Interestingly, the spreading area of neutrophils after LPS stimulation not only correlated with NET rates but also with increasing substrate stiffness [365]. These observations together with the data obtained in manuscript II highlight once more how the cells’

environment can alter the onset of in vitro NETosis and how important standardized assays are for the comparability of in vitro experiments as well as their transferability to in vivo conditions. For instance, distinct tissues reveal different amounts of serum albumin [366], adhesion molecule expression [66] and stiffness [367].

Based on these considerations, it is not surprising that activation with stimuli which do not require receptor-mediated signaling, like PMA, are more stable against modifications of environmental parameters. The same applies for neutrophil adhesion. PMA-induced NETosis progresses mainly independently of Mac-1-mediated adhesion [368] and is functional even in suspended neutrophils derived from patients with leukocyte adhesion deficiency 1 (LAD1;

deficiency in the 2-integrin) [369] and on passivated surfaces (manuscript I, Supp. fig. 12), which severely limit neutrophil adhesion. In direct comparison, PMA-induced NETosis progressed independently of surface passivation, but NETosis was completely impaired in response to LPS [365]. Therefore, the differential requirement of adhesion for NET formation represents an additional explanation for the results presented in manuscript II, namely fully functional PMA-induced NETosis of human neutrophils in serum or serum albumin supplemented media in contrast to LPS (manuscript II, Fig. 1). However, as also reported by Fuchs et al., high serum concentrations (5-20% hiFCS) are sufficient to decrease PMA-induced NETosis even in human neutrophils [98]. One can, therefore, assume that serum proteins still reduce the active PMA concentration or act through other, as yet unidentified, pathways. This provides an additional explanation for the apparent inhibition of PMA-induced NETosis by serum proteins observed for mouse neutrophils (manuscript II, Fig. 3), especially since NET stimulation of murine cells is weak either way.

Apart from direct alteration of NETosis, neutrophils can exhibit an activated or primed phenotype, e.g. in infections and chronic diseases. For instance, neutrophils isolated from patients with diabetes or SLE are more prone to NETosis, presumably owing to their lower activation level for the onset of P1 [69, 70]. In vitro, neutrophil priming was frequently considered for different cytokines such as TNF , which enabled NETosis after IL-8 or LPS activation [76]. Similar mechanisms are conceivable for light-induced NETosis (manuscript III) in vivo. It is unlikely that neutrophils in tissues respond to light with high NET rates under physiological circumstances. However, under pathophysiological conditions, priming of neutrophils possibly decreases the activation level for NETosis particularly in

CHAPTER 4 - Discussion and outlook

Dissertation - Elsa Neubert

induction by light-sensitive substances (manuscript III, Fig. 5) can possibly act additively within the cell and therefore enhance NET rates. Since H2O2 reacts downstream of NADPH oxidase activation within the signaling cascade of ROS-dependent NETosis, it is conceivable that the increasing H2O2 levels not only facilitate or enhance NETosis but also shorten the time of the first phase/P1 and hence accelerate the formation of NETs.

4.1.2 Active modulation of NETosis progression

After stimulation, the progression of NETosis depends on the induction of a specific signaling cascade. Indeed, the duration of P1 varies for different stimuli (manuscript I, Supp. fig. 3).

Not surprisingly and extensively discussed in manuscript I, general alterations of active processes such as temperature variation and energy depletion can profoundly alter the duration of P1 or even entirely abrogate NET formation (manuscript I, Fig. 3). In line with this, even small variations of the neutrophils’ surrounding can interfere with the progression of NETosis. A prominent example is an increase in extracellular pH towards alkaline conditions. In three independent studies, alkaline pH was correlated with enhanced NOX-dependent as well as NOX-inNOX-dependent NETosis. The increased NET rates were linked to enhanced ROS generation (NOX-dependent and NOX-independent), histone 4 cleavage and PAD4-mediated citrullination [370-372]. Therefore, changes in the surrounding conditions not only interfere with the onset of NETosis as discussed in paragraph 4.1.1 (see also manuscript II), they can also directly alter NET progression; an essential observation for studying NETosis in vitro and in vivo.

Specific inhibition of NET-associated enzymes in P1 prevents the further progression of NETosis whereas P2 is not affected as studied by time-resolved MPO inhibition in manuscript I (Fig. 3). Simultaneously to our study (manuscript I), Tatsiy et al. reported a model, which included active modulation of early and late events in NETosis. For all tested inhibitors, they observed a clear inhibition from the beginning on, which became negligible for most inhibitors within the first hour. This observation is in perfect agreement with our data. However, for selected substances, they obtained a sufficient reduction of NOX-dependent NET formation at time-points up to 120 min [224], which would match with our P2. At a first glance, this observation is contradictory to our model. However, the authors use significantly different experimental conditions, which can massively alter the onset and progression of NETosis as already discussed above. Additionally, the quantification of NETosis by extracellular DNA release (PlaNET dye) used in this study, does not allow to draw conclusions regarding chromatin state of the analyzed cells and therefore on how many cells reached the point of no return after 120 min in their setting.

Interestingly, although cells initially spread on the surface (manuscript I, Fig. 1 and 2), adhesion is no prerequisite for PMA-induced NETosis in our model. In contrast, during P1, the cell retracts its body to allow cell rounding and free chromatin swelling within the cell.

One can postulate that the functionality of this cell body retraction is required for the progression of NETosis. In this context, Uotila et al. analyzed the involvement of filamin A

surface expression of integrins, knockout of filamin A enhanced integrin-mediated adhesion and subsequently decreased NETosis in response to LPS and PMA in mice [373]. In agreement with the diminished NETosis rates, enhanced integrin activity was correlated with a decrease of neutrophil elastase (NE) production [373]. Therefore, Uotila et al. suggested that enhanced 2 activity has a negative impact on NETosis [373]. They also proposed a more mechanistic function of filamin A in NET formation because of its actin cross-linking activity and involvement in uropod retraction. Indeed, filamin A depletion may interfere with cell rounding and softening which is, based on our results, a requirement for NET formation within P1. Interestingly, the retraction of the cell membrane from the substrate (manuscript I, Fig. 2, Supp. fig. 4 and Supp. movies 1/2/8/9) is accompanied by significant degradation of the cytoskeleton and stabilization of actin filamentation by jasplakinolide inhibits NETosis persistently and significantly until early P2. In contrast, interaction of actin filament formation with cytochalasin D or latrunculin A can only diminish NETosis in the first half of P1 (manuscript I, Fig. 5). Thus, a functional cytoskeleton seems to be required for early events of NETosis such as translocation of proteins within the cell and most likely active uropod retraction, but the degradation of actin appears to be indispensable for later events such as translocation of NE into the nucleus [228] and further cell softening. Consequently, the cell gets biomechanically prepared for membrane rearrangement, rounding, chromatin decondensation and rupture in P2.