TRPM7 Kinase Is Essential for Neutrophil Recruitment and Function via Regulation of Akt/mTOR Signaling

During inflammation, neutrophils are one of the first responding cells of innate immunity, contributing to a fast clearance of infection and return to homeostasis. However, excessive neutrophil infiltration accelerates unsolicited disproportionate inflammation for instance in autoimmune diseases such as rheumatoid arthritis. The transient-receptor-potential channel-kinase TRPM7 is an essential regulator of immune system homeostasis. Naïve murine T cells with genetic inactivation of the TRPM7 enzyme, due to a point mutation at the active site, are unable to differentiate into pro-inflammatory T cells, whereas regulatory T cells develop normally. Moreover, TRPM7 is vital for lipopolysaccharides (LPS)-induced activation of murine macrophages. Within this study, we show that the channel-kinase TRPM7 is functionally expressed in neutrophils and has an important impact on neutrophil recruitment during inflammation. We find that human neutrophils cannot transmigrate along a CXCL8 chemokine gradient or produce reactive oxygen species in response to gram-negative bacterial lipopolysaccharide LPS, if TRPM7 channel or kinase activity are blocked. Using a recently identified TRPM7 kinase inhibitor, TG100-115, as well as murine neutrophils with genetic ablation of the kinase activity, we confirm the importance of both TRPM7 channel and kinase function in murine neutrophil transmigration and unravel that TRPM7 kinase affects Akt1/mTOR signaling thereby regulating neutrophil transmigration and effector function. Hence, TRPM7 represents an interesting potential target to treat unwanted excessive neutrophil invasion.

During inflammation, neutrophils are one of the first responding cells of innate immunity, contributing to a fast clearance of infection and return to homeostasis. However, excessive neutrophil infiltration accelerates unsolicited disproportionate inflammation for instance in autoimmune diseases such as rheumatoid arthritis. The transient-receptorpotential channel-kinase TRPM7 is an essential regulator of immune system homeostasis. Naïve murine T cells with genetic inactivation of the TRPM7 enzyme, due to a point mutation at the active site, are unable to differentiate into pro-inflammatory T cells, whereas regulatory T cells develop normally. Moreover, TRPM7 is vital for lipopolysaccharides (LPS)-induced activation of murine macrophages. Within this study, we show that the channel-kinase TRPM7 is functionally expressed in neutrophils and has an important impact on neutrophil recruitment during inflammation. We find that human neutrophils cannot transmigrate along a CXCL8 chemokine gradient or produce reactive oxygen species in response to gram-negative bacterial lipopolysaccharide LPS, if TRPM7 channel or kinase activity are blocked. Using a recently identified TRPM7 kinase inhibitor, TG100-115, as well as murine neutrophils with genetic ablation of the kinase activity, we confirm the importance of both TRPM7 channel and kinase function in murine neutrophil transmigration and unravel that TRPM7 kinase affects Akt1/mTOR signaling thereby regulating neutrophil transmigration and effector function. Hence, TRPM7 represents an interesting potential target to treat unwanted excessive neutrophil invasion.

INTRODUCTION
The melastatin-like transient-receptor-potential (TRP) cation channel, TRPM7, is a bifunctional protein fusing a channel pore selective for divalent cations (Mg 2+ , Ca 2+ , Zn 2+ ) to a Cterminal alpha-type serine/threonine kinase (1)(2)(3)(4). TRPM7 channel and kinase activities are interdependent in that Mg 2+ enters through the channel pore and the kinase domain requires Mg 2+ ions for its activity (5). Thus, suppression of the channel activity might also reduce kinase function (6). Known substrates of the TRPM7 kinase include annexin A1, myosin II, Rho A, and SMAD2 (5,7,8). TRPM7 has been proposed to be essential for lymphocyte proliferation and development (9,10). We recently established that TRPM7 kinase activity regulates differentiation of T lymphocytes toward pro-inflammatory T H 17 cells, while anti-inflammatory regulatory T cells remain unaffected (7). Thereby the channel-kinase reins immune system homeostasis (5,7,11). Moreover, TRPM7 is indispensable for lipopolysaccharides (LPS)-induced activation of murine macrophages (12) and has previously been suggested to promote the differentiation of M1 over M2 macrophages (13). The role of TRPM7 in neutrophil function, though, remains unclear. Neutrophils are the most abundant leukocytes in blood and one of the first responding cells during acute inflammation. Therefore, they are essential players in innate immunity (14,15). Following activation, neutrophils migrate toward the inflammatory site, where they eliminate pathogens via phagocytosis, thereby helping to restore tissue homeostasis.
Another key function of neutrophils is the secretion of various cytokines and antimicrobial factors as well as the production of reactive oxygen species (ROS), killing invading pathogens (16). To prevent the spread of the infection, neutrophils are able to release so called neutrophil extracellular traps (NETs), which in turn contribute not only to bacterial clearance but also support blood vessel occlusion and immunothrombosis (17). It is well established that Ca 2+ signaling is pivotal for the recruitment cascade and activation of neutrophils, highlighting the importance of ion channels for neutrophil function (18,19). TRPM7 channel activity has been implicated as a regulator of cell migration by facilitating Ca 2+ oscillations (20)(21)(22). Moreover, TRPM7 was suggested to be involved in neutrophil chemotaxis, adhesion and invasiveness with, yet, rather conflicting results (23)(24)(25). The precise mechanism how TRPM7 might be involved in neutrophil transmigration and activity remains to be explored. Patients with inherited neutrophil deficiencies suffer from severe infections, underscoring the importance of this cell type in immune defense (14). Moreover, unwarranted neutrophil infiltration accelerates tissue damage due to unsolicited inflammation in inflammatory disorders such as multiple sclerosis or rheumatoid arthritis (26,27). Thus, it is critical to gain a better understanding of the role of TRPM7 channel and kinase activities in the signaling cascades triggering neutrophil recruitment.
Using pharmacologic blockade of TRPM7 channel or kinase as well as genetic inhibition of TRPM7 kinase activity, we here resolve the indispensable role of TRPM7 in human and murine neutrophil transmigration and production of reactive oxygen species. We further identify a potential underlying molecular mechanism, with Akt1 as novel downstream target of the TRPM7 kinase.

Mice and In Vivo Experiment
Trpm7tm1.1Mkma C56BL/6 (Trpm7 R/R ) mice (28) were obtained from RIKEN, Japan and housed in single ventilated cages at the animal facility of the Walther Straub Institute of Pharmacology and Toxicology, Ludwig-Maximilians-Universität München, Munich, Germany. All animal experiments were performed in accordance with the EU Animal Welfare Act and were approved by the District Government of Upper Bavaria, Germany, on animal care (permit no. 55.2-1-54−2532-163-2015). Six-to twelve-week-old male and female mice were used for all experiments.

Phagocytosis Assay
Heparinized human whole blood was pre-treated with DMSO, NS8593 (30 µM), TG100-115 (20 µM), or a combination of IPI-549 (160 nM) and nemiralisib (100 nM), respectively for 30 min at 37°C and then incubated with fluorescent Escherichia coli bio particles (pHrodo green E. coli BioParticle Phagocytosis Kit for flow cytometry, Thermo Fisher Scientific) for 30 min at 37°C. Phagocytosis was stopped and cells were fixed according to the manufacturer's protocol. As control, whole blood was incubated with the particles for 30 min at 4°C. Samples were analyzed by flow cytometry (CytoFlex S, Beckmann Coulter) and FlowJo software. CD15 + /CD66b + populations were defined as neutrophils.

Statistical Analysis
All experimental data are presented as mean ± standard error of the mean (s.e.m.). Statistical analyses were performed using GraphPad Prism 8 (GraphPad Software, LLC). Comparisons between two groups were carried out using Student's t-test. Differences between more than two groups were compared either by one-way or two-way analysis of variance (ANOVA), respectively, and Sidak's multiple comparison test was used as a post-hoc test. Each experiment was performed independently at least in triplicate.

TRPM7 Is Essential for Human Neutrophil Chemotaxis and ROS Production
To validate functional expression of TRPM7 in primary human neutrophils isolated from whole blood of healthy donors ( Figure  1A), we subjected CD16 + neutrophils to whole-cell patch-clamp recordings ( Figure 1B, left panel). We confirmed TRPM7-like current activity upon depletion of intracellular magnesium (Mg 2+ ) and Mg-ATP ( Figure 1B, middle and right panel), using our standard internal and external solutions (see Methods). TRPM7 currents were inhibited by the pre-incubation of cells with the known TRPM7 channel inhibitor NS8593 (30 µM) for 15 min, further confirming TRPM7 channel activity in primary human neutrophils ( Figure 1B, red trace). In contrast, the recently described TRPM7 kinase inhibitor, TG100-115 (29) (20 µM, 30 min pre-incubation), did not affect channel activity in our patch-clamp recordings ( Figure 1B, blue trace).
To investigate whether TRPM7 is involved in the regulation of neutrophil function, we first analyzed chemotactic properties of isolated human neutrophils in a transwell assay in response to saline ( Figure 1C, Ctrl, left panels) or the chemokine interleukin 8 (CXCL8 (30,31),) ( Figure 1C, middle panel, 10 nM, 45 min) by flow cytometry. Inhibition of TRPM7 channel activity, using NS8593 (30 µM, 30 min pre-incubation), resulted in significantly reduced neutrophil numbers migrating toward a CXCL8 gradient compared to controls ( Figure 1C, right panel).
Another important physiological function of neutrophils is the killing of invading pathogens via phagocytosis and reactive oxygen burst. In order to elucidate whether this functionality is also affected by TRPM7 blockade, we evaluated phagocytic activity and ROS production of human neutrophils in the presence of TRPM7 inhibitors. To test for phagocytic activity, we incubated human whole blood from healthy donors with fluorescent E. coli particles and analyzed phagocytosis by flow cytometry. While phagocytosis was not affected by TRPM7 channel blockade (NS8593, 30 µM) or kinase inhibition using TG100-115 (20 µM, blue), or a combination of IPI-549 and nemiralisib (IPI/NEM, 160/100 nM, gray) (Figure 2A), we found differences in ROS production in response to the bacterial lipopolysaccharid LPS. TRPM7 channel blockade using NS8593 (30 µM) and TRPM7 kinase blockade using TG100-115 (20 µM) resulted in significantly less ROS production upon stimulation in human neutrophils compared to control cells ( Figure 2B). Comparable to chemotaxis, ROS production was also affected in neutrophils by the inhibition of PI3K-g and -d isoforms demonstrating that TRPM7 and PI3K-g and -d isoforms mediate similar neutrophil functions suggesting that TRPM7 and PI3K-g and -d isoforms might follow analogous or even sequential signaling pathways.

TRPM7 Regulates Human Neutrophil Function via NFkB, Erk, and Akt/mTOR Signaling Pathways
To further unravel the mechanism and signaling pathways by which TRPM7 channel and/or kinase blockade alters neutrophil function, we analyzed the effect of the different TRPM7 moieties on cellular signaling. Typically, chemotaxis and migration of neutrophils is regulated by LPS-triggered pathways followed by NFkB activation (32,33). For cell motility and chemotaxis also Erk1-dependent signaling cascades are essential (34). Aside of NFkB and Erk1, PI3K/Akt/mTor signaling pathways regulate neutrophil function and recruitment (35,36). Therefore, we employed a bead-based Bio-Plex assay (Bio-Rad) to simultaneously measure the phosphorylation status of multiple signaling proteins in the same sample (37). We examined phosphorylation levels of NFkB (p65 Ser 536 ), ERK1/2 (Thr 202 / Tyr 204 , Thr 185 /Tyr 187 ), Akt (Ser 473 ), and mTOR (Ser 2448 ) in LPS stimulated primary human neutrophils ( Figures 2C-F). TRPM7 channel blockade using NS8593 (30 µM) revealed a reduction in NFkB, ERK1, and Akt/mTOR-dependent signaling, whereas TRPM7 kinase blockade using TG100-115 (20 µM) only led to a reduction in Akt-dependent signaling. Interestingly, inhibition of PI3K-g and -d using IPI-549 and Nemiralisib (IPI/NEM, 160 nM/100 nM) did not show any impact ( Figures 2C-F) on the phosphorylation status of the detected proteins in human neutrophils. This indicates that TRPM7 might not directly signal through activation of PI3K-g and -d in human neutrophils or that other isoforms can compensate, at least for the investigated downstream signaling molecules. The impact of PI3K-g and -d blockade on chemotaxis and ROS production might not involve the analyzed signaling targets and thus might not be compensated for.

TRPM7 Kinase Is Essential for Neutrophil Transmigration and Infiltration in an In Vivo Peritonitis Model
To further elucidate the role of TRPM7 kinase activity for neutrophil function, we next analyzed whether genetic inactivation of the TRPM7 kinase conveys similar effects on neutrophil chemotaxis and transmigration as pharmacologic blockade by TG100-115. We took advantage of a mouse model carrying a point mutation at the active site of the enzyme. Mutating lysine at position 1646 to arginine (Trpm7 R/R ) disrupts ATP binding and thereby kinase activity (28). We have previously shown, that this mutation affectively abolishes phosphotransferase activity, while leaving the channel function of TRPM7 intact (7). Here, analogously to human neutophils, we subjected CD16 + bone marrow derived murine neutrophils ( Figure 3A) from Trpm7 +/+ and Trpm7 R/R mice to whole-cell patch-clamp recordings ( Figure 3B). We confirmed TRPM7-like current activity upon depletion of intracellular Mg 2+ and Mg-ATP ( Figure 3B, middle and right panel), using our standard internal and external solutions (see Methods). TRPM7-like currents were blocked by the application of the known TRPM7 channel inhibitor NS8593 (30 µM, pre-incubation for 15 min), further confirming TRPM7 channel activity in primary murine neutrophils ( Figure 3B, red traces). Thus, our electrophysiologic analysis established the functional expression of TRPM7 on murine bone marrow derived neutrophils, with similar current development and amplitude as well as inhibition in response to NS8593 between the two genotypes ( Figure 3B). This further indicates that in neutrophils the channel function is independent of its kinase activity.
To further delineate whether neutrophil transmigration is regulated by TRPM7 channel and/or kinase activities, we perfomed a transmigration-assay using primary bone marrowderived neutrophils isolated from TRPM7 kinase-deficient (Trpm7 R/R ) and recpective wild-type (TRPM7 +/+ ) mice. We analyzed chemotactic properties of murine neutrophils in a transwell assay ( Figure 3C) in response to saline or CXCL1 (keratinocytes-derived chemokine, KC), the murine counterpart to CXCL8 ( Figure 3C, left panel, 10 nM, 45 min) by flow cytometry. Inhibition of TRPM7 channel activity, using NS8593 (30 µM, 30 min pre-incubation), resulted in reduced Trpm7 +/+ neutrophil numbers migrating toward a CXCL1 gradient compared to controls, albeit not significantly ( Figure  3C, right panel). Interestingly, TRPM7 kinase-deficient Trpm7 R/R neutrophils ensued a significant reduction in neutrophil CXCL1triggered chemotaxis. Additional inhibition of TRPM7 channel activity using NS8593 even further reduced the numbers of migrating Trpm7 R/R neutrophils in response to CXCL1. These results suggest that both TRPM7 kinase and channel activity contribute to chemotaxis of murine neutrophils.
In order to apprehend whether circulating neutrophils or other leukocyte numbers were different in Trpm7 R/R mice compared to Trpm7 +/+ , we analyzed the distribution of white blood cells in whole blood performing a differential blood count via a hematology analyzer (IDEXX ProCyte Dx). Notably, white blood cell counts were similar between Trpm7 R/R mice and Trpm7 +/+ controls ( Figure S1A), indicating no major differences in the composition of circulating leukocytes. To finally understand the impact of TRPM7 kinase moiety on the function of neutrophils in vivo, we employed a TNF-a-induced peritonitis model using TRPM7 kinase-deficient (Trpm7 R/R ) mice. The intraperitoneal application of TNF-a triggers the recruitment of circulating leukocytes to sites of inflammation through upregulation of endothelial-specific adhesion relevant molecules (38). We pre-treated Trpm7 +/+ or Trpm7 R/R mice with saline only (Ctrl) or TNF-a (500 ng) intraperitoneally (i.p.), respectively. Four hours post injection, we analyzed neutrophil infiltration into the peritoneal cavity via flow cytometry ( Figure  3D, left panels). In Trpm7 +/+ mice, i.p. injection of TNF-a dramatically increased the number of neutrophils in the peritoneal cavity compared to control mice ( Figure 3D, right panels). In contrast, neutrophils from Trpm7 R/R mice showed a dramatic reduction in neutrophil transmigration into the peritoneal cavity upon TNF-a stimulation ( Figure 3D), suggesting a critical role of TRPM7 kinase in this process.
Notably, the application of the channel blocker NS8593 resulted in a similar LPS-induced phosphorylation status compared to Trpm7 R/R controls for all signaling proteins, further suggesting that TRPM7 channel blockade might also affect kinase activity.
To confirm that the effects of pharmacologic inhibition of the TRPM7 channel or kinase using NS8593 or TG100-115 on the respective signaling proteins was not due to off-target effects, we incubated Trpm7 R/R neutrophils for 30 min with NS8593 or TG100-115 prior to LPS stimulation. Indeed, the pharmacologic blockade of TRPM7 kinase by TG100-115 in Trpm7 R/R neutrophils did not further reduce the phosphorylation status of NFkB (p65 Ser 536 ), ERK1/2 (Thr 202 /Tyr 204 , Thr 185 /Tyr 187 ), Akt (Ser 473 ), or mTOR (Ser 2448 ) ( Figures 4E-H) compared to Trpm7 R/R controls. Similar results were found for application of the channel blocker NS8593 (Figures 4E-H).
Together our findings indicate that the TRPM7 kinase regulates neutrophil function via activation of Akt/mTOR pathways, while channel activity controls neutrophil activity also through NFkB-dependent pathways, most likely due to providing essential calcium and magnesium ( Figure 4I).

DISCUSSION
Excessive neutrophil infiltration accelerates tissue damage due to unrestrained inflammation in pro-inflammatory as well as autoinnmue diseases (26,27). Therfore, it is crucial to understand the mechanisms that regulate neutrophil function and migration. Previously, TRPM7 was suggested to be involved in CD147-triggered Ca 2+ -induced chemotaxis, adhesion, and invasiveness of a human neutrophil cell line as silencing TRPM7 via siRNA led to a reduction of neutrophil adhesion (23). However, the precise mechanism how TRPM7 might be involved in neutrophil recruitment still remains to be explored. Utilizing a heterozygous TRPM7 kinase-deficient mouse model, Trpm7 +/DK (39), it was shown that channel and or kinase activity might affect neutrophil rolling and recruitment (25). Consequently, authors concluded that the channel-kinase TRPM7 could have a protective role in cardiovascular inflammation and fibrosis (25). As in the respective model also the channel activity is reduced (39,40) it is critical to gain a better understanding of the role of TRPM7 channel and kinase activities in the signaling cascades triggering neutrophil recruitment. We here show that pharmacologic and genetic inhibition of TRPM7 kinase affects human and murine neutrophil function. We further identify a potential underlying molecular mechanism, with Akt1 as novel downstream target of the TRPM7 kinase in neutrophils.
Along with other ion channels, TRPM7 has been linked to migration and motility of various different cell types (41). It has been suggested that TRPM7 channel activity is aiding calcium (Ca 2+ ) flickers thus steering fibroblast cell migration (22). We here propose, that in addition to facilitating Ca 2+ or Mg 2+ influx, TRPM7 might affect migration and motility via its kinase domain. TRPM7 might do so via direct phosphorylation of myosin II (29,42). Recently, a compound with TRPM7 kinase inhibitory activity was identified in a small molecule library screen. TG100-115 was the most potent inhibitor in the screen, significantly decreasing cell migration and invasion of breast cancer cells and inhibiting TRPM7 kinase-dependent myosin IIA phosphorylation (29). Although TG100-115 has been reported to suppress TRPM7 ion channel activity (29), in our hands the kinase inhibitor had no effect on ion channel function. Originally, TG100-115 was shown to inhibit PI3K-g and -d (43), to which its broad anti-inflammatory and anti-cancerous as well as cardio-protective effects were attributed (43,44). However, at least in part, this could also be due to its TRPM7 kinase inhibition. To assess whether the observed effects of | TRPM7 regulates neutrophil function via NFkB and Akt/mTOR signaling pathways. Assessment of the activity of the cell signaling molecules NFkB, Erk1/2, Akt1, and mTOR utilizing a Bio-Plex assay and phospho-specific antibodies on lysates of bone marrow derived murine neutrophils of Trpm7 +/+ (black) and Trpm7 R/R (green) mice. Trpm7 +/+ and Trpm7 R/R neutrophils were pre-incubated with or without (control, black) the TRPM7 inhibitor NS8593 (30 µM, red), the TRPM7 kinase blocker TG100-115 (20 µM, blue), or a combination of IPI and NEM (160 and 100 nM, gray), respectively, for 30 min. Presented data depict the phosphorylation status upon stimulation with 10 ng/ml LPS for 30 min of (A, E) NFkB p65 (Ser536), (B, F) Erk1/2 (Thr202/Tyr204, Thr185/Tyr187), (C, G) Akt (Ser473), and (D, H) mTOR (Ser2448). For comparison results from control Trpm7 R/R neutrophils (open green triangle) were taken from the respective panels above. Data are normalized to protein content and represented as mean ± s.e.m.; n = 3, measured in duplicates; a total number of 5-6 mice were used for each genotype. Statistics: one-way ANOVA *p < 0.05, **p < 0.01, ***p < 0.005. (I) Molecular model illustrating the role of TRPM7 in neutrophil chemotaxis and function: in macrophages, LPS-dependent endocytosis of TLR4 and subsequent NFkB activation was shown to depend on TRPM7 (12). We confirm a similar pathway involving TRPM7 channel activity in neutrophils (red dashed arrow). Previously, TRPM7 channel moiety was suggested to be positioned alongside the PI3K signaling axis (red dashed arrow). In neutrophils, PI3K isoforms are activated via CXCL8 and CXCR1/2, further triggering the activation of Akt1, NFkB, and mTOR signaling cascades. Alternatively, we propose a model in which TRPM7 kinase is directly or indirectly phosphorylating Akt1, thereby controlling NFkB and mTOR signaling pathways (blue dashed arrow).
TG100-115 are primarily due to PI3K-g/d blockade, we applied distinct PI3K-g/d inhibitors (IPI, NEM) with similar IC 50 values (43). While affecting neutrophil chemotaxis in response to a CXCL8 gradient, as well as the production of reactive oxygen species (ROS) upon stimulation with the gram-negative bacterial lipopolysaccharide LPS, PI3K-g/d inhibitors did not decrease the LPS-triggered phosphorylation of NFkB, ERK1/2, Akt1, or mTOR. Blockade of TRPM7 kinase using TG100-115, however, affected neutrophil chemotaxis as well as ROS production in response to LPS most likely due to a reduction in Akt/mTOR signaling pathways. Interestingly, TRPM7 inhibition with the channel blocker NS8593 resulted in a similar reduction of Akt/mTOR signaling pathways, culminating in diminished neutrophil chemotaxis and ROS production. It is therefore tempting to speculate that TRPM7 channel blockade also reduces kinase activity and that the Mg 2+ entering through the channel is required for optimal kinase function. TRPM7 kinasedeficient murine neutrophils also displayed reduced chemotaxis toward a CXCL1 gradient, which was further decreased upon TRPM7 channel blockade using NS8593, suggesting that TRPM7 channel function, at least in part, acts independent of TRPM7 kinase due to ion conductance. Previously, TRPM7 activity has already been implicated alongside PI3 kinase signaling pathways (10,45,46), however, as of now the impact of TRPM7 channel versus kinase moiety on different PI3K isoforms remains to be established. We here used PI3K isoform specific inhibitors as well as genetic inactivation of TRPM7 kinase to shed light on their interrelationship. In human LPS-treated neutrophils downstream analysis of signaling targets did not reveal a direct functional connection between TRPM7 and PI3K-g/d. However, functional analyses of neutrophil chemotaxis and ROS production indicated a potential interdependence. One possible explanation could be that, in CXCL1-triggered chemotaxis as well as LPS-induced ROS production, signaling molecules depending on PI3K-g/d pathways cannot be remunerated for, while the LPS-dependent phosphorylation of NFkB, Erk1/2, Akt1, and mTOR was compensated by other PI3K isoforms. Accordingly, TRPM7 kinase might directly or indirectly phosphorylate Akt1 and thereby control complex PI3 kinase signaling pathways while the channel function may indirectly act on PI3 kinase signaling via providing essential Ca 2+ and/or Mg 2+ for proper kinase activity ( Figure 4I). Taken together, we have shown that TRPM7 regulates several neutrophil effector functions including transmigration, extravasation, and ROS production. In addition, we were able to determine that TRPM7 kinase activity leads to Akt/mTOR activation, while the TRPM7 channel moiety also mediates activation of NFkB and ERK1/2. Finally, we demonstrate that TRPM7 and PI3K-g/d are not directly linked via those signaling pathways. Additional work is needed to further sort out how TRPM7 regulates neutrophil effector functions and how TRPM7 and PI3K-g/d cooperate in mediating those neutrophil functions. To finally understand whether TPRM7 kinase activity also affects neutrophil transmigration in vivo, we employed a TNFdependent peritonitis model (47). While this short-term model does not exclude potential effects TRPM7 might have on other cell types, including the migration of other immune cells at later stages, it emphasizes the importance of TRPM7 kinase in regulating neutrophil effector functions and highlights the urgent need for more specific pharmacological inhibitors targeting the TRPM7 kinase moiety.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by Ethics committee from the Ludwig-Maximilians-Universität München (Az. 611-15). The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by District Government of Upper Bavaria, Germany. Written informed consent was obtained from the owners for the participation of their animals in this study.