In 2004, a definition was provided for NET that was followed by a massive wave of research on neutrophils that led to emphasizing their importance in acute and chronic inflammation [5, 8]. These studies proved that NET production increases under inflammatory conditions, including in SLE [8]. NET was shown to directly influence inflammation [20] and provide autoantigens for pathologic autoantibodies during SLE [2]. More notably, as a large complex compound in which many types of DAMPs interact with each other, NET can interplay with the immune cells [12,13,14], including its producers, i.e. neutrophils [21]. Nonetheless, there is a serious discrepancy in the role of NET in inflammation [6, 20, 22]; in particular, the interactions of neutrophils with NET have been rarely inspected. The present study evaluated the effect of NET on neutrophils in the context of SLE.
Regarding the prominence of cell mortality in SLE [2], the viability and apoptosis of neutrophils upon encounter with normal and SLE neutrophil-derived NET were assessed; while NET augmented the percentage of late apoptotic cells, it did not affect the early apoptotic cells of neutrophils. Although some studies have reported that NET or its components induce apoptosis in some cell types [23], it cannot be deduced from the present findings that NET-treated neutrophils undergo apoptosis more than unaffected granulocytes because of the unchanged rate of early apoptosis.
To explain the increase in late apoptotic neutrophils without a raise in the early apoptotic cells, it should be noted that every dying cell with asymmetric and permeable plasma membrane that represents the annexin+/PI+ phenotype after annexin/PI double staining is not necessarily a late apoptotic cell. The externalization of phosphatidylserine (PS) and permeabilization of the plasma membrane are also seen in other types of cell death, particularly in pyroptosis and NETosis [24]. It is thus highly likely that some of the non-apoptotic dying cells, especially NETting and pyroptotic cells, are wrongly considered as the late apoptotic cells in studies that evaluated PS expression plus the plasma membrane integrity to assess apoptosis; and, as such, in the present study [25]. From this point of view, the results of the current study suggest that NET increases cell demise by the augmentation of cell death modes other than apoptosis –more likely, pyroptosis and NETosis. This conclusion clearly needs further proof by well-designed studies; however, there is already ample evidence for this deduction:
First, the percentage of PS-externalizing neutrophils with an intact membrane (annexin+/PI−), which are considered specific features of (early) apoptotic cells [24], was not affected by NET. Second, studies have reported an enhanced NETosis in human neutrophils by HMGB1 and extracellular histones, which collectively constitute more than 70% of NET-associated proteins [6, 26], and also by NET-containing media collected from PMA-stimulated neutrophil cultures [21, 27]. Third, several studies on different DAMPs, such as HMGB1 [28] and histone [29] and even the whole NET [29], have established that these DAMPs can induce pyroptosis in different types of cells [30, 31]. Similarly, Kahlenberg et al. [15] showed that the NET isolated from both the lupus and control subjects was an effective activator of pyroptosis in human macrophages. More relevantly, Chen et al. [32] demonstrated that the NET collected from PMA-stimulated PMN following coculture with normal bone marrow-derived macrophages caused cell pyroptosis. Nevertheless, neither of these or other studies have explored pyroptosis in human granulocytes during SLE, but the contribution of the DAMP-induced pyroptosis of different types of cells in the pathology of SLE has been demonstrated in a large number of reports [15, 33]; therefore, the increase in the pyroptosis of granulocytes in response to the DAMP carrier structure of NET is well conceivable. Fourth, the NET obtained from SLE patients, which is expected to carry a higher level of DAMP [9], resulted in a greater increase in the annexin+/PI+ cell population than the NET obtained from healthy subjects.
The present findings also showed a rise in the necrosis of NET-treated neutrophils, which is compatible with previous studies that had demonstrated that various DAMPs, including nucleosomes, histone, and entire NET, can induce necrosis in multiple types of immune or non-immune cells [21, 29, 34]. Also, the greater increase in the percentage of necrotic neutrophils following incubation with SLE NET compared to those incubated with healthy NET can be explained by the higher content of DAMPs in SLE NET [9, 35].
Given our results and other above-mentioned studies, the decreased number of viable neutrophils observed in the current study after 4 h of co-incubation with NET is not due to the increased apoptosis, which is considered the only kind of cell death of an anti-inflammatory nature [33], but reflects the augmentation of necrosis and potentially other inflammatory cell-death modalities, most likely, NETosis and pyroptosis [25]. Of course, this notion should be confirmed by further studies that would directly measure other types of cell death.
The upregulation of CD11b expression in neutrophils following NET exposure was assessed as an activation/degranulation marker of granulocytes [8]. The present findings revealed an obvious increase in the expression of CD11b on LPS-stimulated granulocytes after encounter with NET. The review of literature did not yield any studies on the effect of whole NET on CD11b expression on granulocytes; however, studies on separate DAMPs have shown an upregulation of CD11b on neutrophils after stimulation by nucleosome, Myeloperoxidase (MPO) and Calprotectin (S100A8 and S100A9) [36,37,38] –all of which are important constituents of NET [7, 26]. Moreover, an additional increase was observed following incubation with SLE NET compared to those incubated with healthy NET, which is in agreement with the reports by Ribon et al. [39], who showed a higher upregulated CD11b expression on neutrophils following exposure to higher levels of purified chromatin, or the study by Lindau et al. [40], who demonstrated a clear dose-dependent effect for nucleosomes on the upregulation of CD11b expression in neutrophils.
Despite the sufficient number of studies on the contribution of ROS and their induced oxidative stress in the pathology of SLE [1, 3], the impact of whole NET or its many well-known contributing DAMPs, such as histones, nucleosome, and chromatin, on granulocytes’ oxidative burst (OB) remains exceptionally unexplored. Likewise, few investigations were found on the effect of NET-related DAMPs on the OB of neutrophils. Sroussi et al. [41] showed that Calprotectin (S100A8 and S100A9) inhibited the OB of neutrophils. Lau et al. [36] found that MPO increases the ability of OB in human neutrophils. Tadié et al. [42] observed that the exposure of human neutrophils to HMGB1 diminishes the rate of OB. All of these DAMPs are fundamental elements of NET [6, 26]; this fact, along with the lack of data about other NET-associated DAMPs, make explaining the impact of NET on the respiratory burst of neutrophils a very intricate matter. Nonetheless, Tadié et al. [42] found the salient point that two distinct DAMPs (HMGB1 and S100B, both key components of NET) binding to the same PRR inversely affect the rate of neutrophil OB. Moreover, the same study [42] showed that different DAMPs interfere with each other’s effect on the OB of neutrophils. Similar functions also can be expected from NET as a rich source of DAMPs; although each contributing component of NET may affect the OB ability of neutrophils individually, the overall effect derives from the whole existing DAMPs in NET, and their interfering with each other is to potentiate the OB capability of neutrophils, as shown here.
One of the limitations of the present research was that the exact components of the two NET types were not compared. Thus, the responsible agent/s for more enhancements of cell mortality, CD11b expression, and OB ability of neutrophils in SLE NET could not be identified. The work of Bruschi et al. [43], who recently investigated differences between the protein composition of SLE and normal neutrophil-derived NET, may help propose scientific suggestions in this regard. They demonstrated a notable increase in the level of HMGB1 and histone H1, a slight increase in Calprotectin, and a notable decrease in the level of MPO in SLE NET compared to normal NET. Although these changes may be the causes of additional increment of mortality [27, 31] and CD11b expression [37, 44], they do not explain the increased OB capacity of neutrophils. They also found several proteins that were differently expressed by the two NET types, the impacts of which on neutrophil functions have not been elucidated yet. Regarding these issues plus the fact that the interaction of DAMPs with each other can change their effects [42, 45], determining the exact component(s) which mediate the extra effect of SLE NET need to be explored in the future, well-designed inhibitory investigations.
This study was conducted to investigate how, as a biological product enriched by multiple DAMPs [7], NET influences neutrophil effector responses and whether the NET produced by the neutrophils of SLE patients differs from healthy neutrophil-derived NET in terms of affecting neutrophil activities. According to the present findings, NET diminished the longevity of neutrophils and hastened their death. While this phenomenon may help alleviate ongoing inflammation by the elimination of neutrophils as important inflammatory cells [5, 6], the pro-inflammatory death (necrosis and potentially pyroptosis and NETosis) induced by NET can further intensify the inflammation. Moreover, it increases the burden of dead cells and thus exacerbates the disease.
Furthermore, DAMPs incorporated within NET cause neutrophils to increase CD11b upregulation. Regarding the key role of CD11b in most inflammatory activities of granulocytes, such as degranulation, neutrophil recruitment, and aggregation at inflammatory sites [8], these neutrophils can more actively contribute to the ongoing inflammation and aggravate the disease. However, recent researches suggest two anti-inflammatory functions for CD11b in neutrophils during SLE [46].
Since ROS can act as a double-edged sword in SLE pathogenesis [35], the effects observed for NET on the respiratory burst of neutrophils cannot be easily interpreted. Surplus ROS production can result in enhanced oxidative stress, which is involved in SLE pathogenesis [3]. Alternatively, the deficiency of ROS allows for the excessive degranulation of neutrophils following stimulation, leading to tissue damage and thereby sustaining inflammation via the release of DAMPs; also, the impairment of ROS can increase predisposition to SLE [35, 47].