In addition to bacterial, fungal, parasitic and viral pathogens, host-derived endogenous inflammatory mediators such as crystals, cytokines, chemokines, immune complexes and autoantibodies and a plethora of further agents are potent inducers of NET formation[17–21]. Previous studies have shown that MSU-crystals induced NET formation is preceded by elevated cytoplasmic calcium levels and, consequently, by the generation of ROS[16, 22]. As important intracellular messenger, high cytosolic Ca2+ levels increase the activity of protein kinase C (PKC) which phosphorylates gp91phox. The latter activates NADPH oxidase to generate ROS and thus initiates the downstream signaling that finally results in oxidative burst and NET formation. Furthermore, high cytosolic Ca2+ levels activate PAD4, cause histone citrullination and thus promote chromatin depolymerization[23, 24]. However, the mechanisms by which MSU crystals trigger NET formation are still elusive.
Here, we show that TRPM2, a nonselective cation channel, mediated MSU crystals- induced NET formation by regulating calcium influx and ROS production. Deletion of TRPM2 impairs the capacity of neutrophils to form NETs and aggNETs in response to MSU crystals; TRPM2-/- neutrophils displayed reduce cytoplasmic calcium levels and ROS production. This is consistent with the mechanism described for H2O2-mediated NETs formation[25]. Deletion of TRPM2 protects against the inflammation caused by MSU crystals.
MSU crystals, one of the prototypic endogenous DAMPs, act as danger signal and elicit robust inflammation[26]. After uptake of MSU crystals, mononuclear phagocytes like monocytes and macrophages engage the caspase-1-activting NALP3 inflammasome[27] to process pro-IL1β and to generate the active form of the proinflammatory cytokine IL-1β, which consequently triggers a feverish inflammation and recruits further neutrophils[7]. The latter vigorously ingest MSU crystals and release large amounts of pro-inflammatory cytokines and chemokines, including TNF-α and IL-6. This precipitates the acute inflammatory reactions and initiates the activation of the adaptive immune response[28]. MSU crystals-induced NET formation also contributes to the resolution of the inflammatory process. Indeed aggNETs promoted the resolution of the MSU crystal-induced inflammation by promoting the proteolytic degradation of inflammatory cytokines and chemokines.
In Ncf1** mice that show an impaired NOX2-dependent generation of ROS and a reduced formation of aggNETs, the neutrophil-driven inflammation in response to MSU crystals exacerbated and became chronic[8]. Here, in MSU crystals-induced murine air pouch inflammation, TRPM2-/- mice showed reduced infiltration of inflammatory cells as well as reduced concentrations of pro-inflammatory mediators in their air pouches. Similar results were to be observed in the neutrophil-driven, MSU crystal-induced paw edema model. Again, the inflammation was alleviated in TRPM2-/- mice.
Previous studies showed that although deficient NET formation delayed the inflammatory responses it also abrogated the resolution of inflammation. In contrast, TRPM2-/- mice that preserved some ROS production and NETs formation showed an overall alleviation of the MSU crystal-induced inflammation with intact resolution. Comparing our results to those of older studies, it must be pointed out that Ncf1** mice which carry a single mutation of the neutrophil cytosolic factor 1 (encoded by Ncf1), completely lose the ability for NOX2- dependent ROS generation. In this study, TRPM2-/- mice that have been generated by the deletion of the third and fourth transmembrane domains of the channel protein, produce some ROS and still form (reduced numbers of) NETs. Importantly, these NETs differ morphologically from those of wild type mice.
TRPM2 is highly expressed in many kinds of immune cells [29]. Thus, the deletion of TRPM2 in other immune cells, e.g. macrophages may indirectly affect neutrophils as TRPM2 is involved in the cytokines production of mononuclear phagocytes[30]. Published studies on the role of TRPM2 in neutrophils are quite conflicting. This is an example of the subtle balance the innate immune system needs to cope with the plethora of potential invaders that must reliably be identified and ignored or fended off. Depending on the types of disease models under investigation, TRPM2 deletion consequently leads to an increased[31] or decreased[9, 32–34] production of pro-inflammatory cytokines. However, most studies consider TRPM2 a potential therapeutic target for antagonizing oxidative stress-related pathological conditions such as diabetes, inflammation, neurodegeneration, cardiovascular disease, and stroke[11, 35]. Accordingly, we propose that TRPM2-/- mice alleviate neutrophil-driven inflammation by a combination of altered NET function and reduced release of inflammatory mediators.