The alive and accommodating action of deepening resolution is ytical for tissue homeostasis, and abnormal resolution is associated with abiding anarchic diseases. Specialized proresolving mediators (SPMs) are blubbery acerbic derivatives that annul the furnishings of proinflammatory factors, triggering resolution and tissue repair. SPM biosynthesis depends on the cytosolic localization of the nonphosphorylated anatomy of the lipoxygenase 5-LOX, admitting the nuclear about-face of phosphorylated 5-LOX after-effects in anarchic leukotriene production. Cai et al. showed that signaling by the efferocytosis receptor MerTK in macrophages activated an ERK-dependent alleyway that inhibited the phosphorylation of 5-LOX, appropriately announcement SPM production. Inhibition of ERK activation in a abrasion archetypal of peritonitis delayed resolution, suggesting that the MerTK-ERK alleyway adeptness be therapeutically manipulated to advance deepening resolution.
Inflammation resolution counterbalances boundless deepening and restores tissue homeostasis afterwards injury. Failure of resolution contributes to the anatomy of abounding abiding anarchic diseases. Resolution is advised by autogenous specialized proresolving mediators (SPMs), which are acquired from long-chain blubbery acids by lipoxygenase (LOX) enzymes. 5-LOX plays a ytical role in the biosynthesis of two classes of SPMs: lipoxins and resolvins. Cytoplasmic localization of the nonphosphorylated anatomy of 5-LOX is capital for SPM biosynthesis, admitting nuclear localization of phosphorylated 5-LOX promotes proinflammatory leukotriene production. We ahead showed that MerTK, an efferocytosis receptor on macrophages, promotes SPM biosynthesis by accretion the affluence of nonphosphorylated, cytoplasmic 5-LOX. We now appearance that activation of MerTK in beastly macrophages led to ERK-mediated announcement of the gene encoding sarcoplasmic/endoplasmic cloth calcium ATPase 2 (SERCA2), which decreased the cytosolic Ca2 absorption and suppressed the action of calcium/calmodulin-dependent protein kinase II (CaMKII). This, in turn, bargain the activities of the mitogen-activated protein kinase (MAPK) p38 and the kinase MK2, constant in the added affluence of the nonphosphorylated, cytoplasmic anatomy of 5-LOX and added SPM biosynthesis. In a zymosan-induced peritonitis model, an anarchic ambience in which macrophage MerTK activation promotes resolution, inhibition of ERK activation delayed resolution, which was characterized by an added cardinal of neutrophils and decreased amounts of SPMs in tissue exudates. These allegation accord to our compassionate of how MerTK signaling induces 5-LOX–derived SPM biosynthesis and advance a ameliorative action to accession deepening resolution in settings area abnormal resolution promotes ache progression.
Timely resolution responses afterwards astute deepening are capital for tissue homeostasis (1, 2), and broken resolution is the basal account of assorted abiding anarchic diseases, including cardiovascular disease, anarchic bowel disease, assorted sclerosis, arthritis, and asthma (3–7). Deepening resolution is an active, awful accommodating action that is controlled by assorted autogenous specialized proresolving mediators (SPMs), such as lipoxins, resolvins, protectins, and maresins (1, 8, 9). SPMs amend proinflammatory mediators during astute deepening and actuate resolution by blocking neutrophil infiltration, acceptable the approval of asleep beef (efferocytosis), and acclimation tissue accident afterwards compromising host aegis (1, 10).
SPMs are actinic by lipoxygenase (LOX) enzymes from long-chain blubbery acids, such as arachidonic acerbic (AA), which is appear from phospholipids by the action of cytosolic phospholipase A2; docosahexaenoic acerbic (DHA); and eicosapentaenoic acerbic (EPA) (11). We and others accept apparent that the intracellular localization of 5-LOX determines whether it stimulates the assembly of proinflammatory leukotrienes or proresolving lipoxins. Nuclear 5-LOX, because of its adjacency to leukotriene A4 (LTA4) hydrolase, leads to the about-face of AA to leukotrienes (LTB4) in macrophages (12–14). In contrast, cytoplasmic 5-LOX, because of its adjacency to 12/15-LOX, which is the murine ortholog of beastly 15-LOX and has both 12-LOX and 15-LOX activities (15–17), promotes the about-face of AA to lipoxins (LXA4) or DHA to resolvins (RvD1) (14). The nuclear localization of 5-LOX is advised by its phosphorylation at Ser271 by mitogen-activated protein kinase (MAPK)–activated protein kinase 2 (MK2), which is afterwards of the MAPK p38 (13, 18). Antecedent assignment showed that a p38-MK2 alleyway is activated by cytosolic Ca2 -dependent calcium/calmodulin-dependent protein kinase II (CaMKII) (14, 19, 20), and we showed ahead that Ca2 -mediated CaMKII activation in macrophages increases the affluence of proinflammatory LTB4 and decreases the bulk of proresolving LXA4 (14). However, the upstream regulators of CaMKII in this 5-LOX–derived SPM biosynthesis alleyway abide unknown.
MerTK, a affiliate of the Tyro-Axl-MerTK (TAM) ancestors of receptor tyrosine kinases, is a macrophage receptor that mediates the bounden and phagocytosis of apoptotic cells, a action accepted as efferocytosis. MerTK interacts with apoptotic beef through the bridging molecules Gas6 or protein S, which bind to MerTK on macrophages and to embodied phosphatidylserine on apoptotic beef (21, 22). In accession to mediating efferocytosis, assurance of MerTK by apoptotic cells, Gas6, or protein S triggers two chip but biochemically audible responses: anti-inflammation and proresolution. Accident of these responses by abiogenetic targeting of MerTK in mice can advance to abiding diseases of deepening and broken resolution, conspicuously a lupus-like ache in beforehand mice and atherosclerosis in hypercholesterolemic mice (23–26). The anti-inflammatory acknowledgment involves a alleyway that leads to the aishment of nuclear agency κB (NF-κB)–mediated signaling (27, 28). Affirmation for a audible proresolution acknowledgment was appear by studies application both able macrophages and assorted in vivo models (29, 30). For example, we approved that MerTK signaling in macrophages and in abrasion models of antiseptic peritonitis and ischemia-reperfusion abrasion promotes SPM biosynthesis and enhances resolution by abbreviating the bulk of 5-LOX phosphorylated at Ser271 (p–Ser271–5-LOX) and accretion the affluence of cytoplasmic 5-LOX (30). Moreover, the ectodomain of MerTK can be broken by the protease ADAM metallopeptidase area 17 (ADAM17), and in mice harboring a genetically engineered anatomy of MerTK that is cleavage-resistant (MertkCR), SPM biosynthesis and deepening resolution are enhanced, and atherosclerosis progression is suppressed (26, 30).
The algid of this abstraction was to abode a ytical gap in this field, namely, the apparatus by which MerTK activation triggers resolution signaling in macrophages. Added specifically, we approved to accept how assurance of MerTK increases the affluence of nonphosphorylated, cytoplasmic 5-LOX and the consecutive biosynthesis of LXA4 and RvD1. We showed that the Gas6-MerTK alleyway suppressed the aloft Ca2 –CaMKII–p38–MK2–p–5-LOX signaling alleyway by abbreviating the absorption of cytosolic Ca2 . Cytosolic Ca2 affluence was decreased through MerTK-mediated activation of the MAPK extracellular signal–regulated kinase (ERK), which, in turn, angry the action of SERCA, the calcium ATPase that pumps cytoplasmic Ca2 aback into the endoplasmic reticulum.
RvD1 increases the affluence of nonphosphorylated, cytoplasmic 5-LOX and the assembly of LXA4 in macrophages by suppressing the CaMKII–p38–MK2 avalanche (14). Given that MerTK activation additionally affects 5-LOX and LXA4 in a agnate address (30), we advised the aftereffect of MerTK activation on these three kinases. Beastly monocyte–derived macrophages (referred to as “human macrophages” throughout the study) were advised with MERTK-specific baby interfering RNA (siRNA) or accolade ascendancy siRNA and again incubated with conditioned average from HEK 293 beef cogent the MerTK activator Gas6 or ascendancy conditioned medium. Corpuscle extracts were again yzed by immunoblot for the phosphorylated forms of the three kinases as a admeasurement of their activation. Constant with our antecedent allegation from abrasion macrophages (30), appraisal of beastly macrophages with Gas6 added the affluence of p-MerTK (Fig. 1A). As was the case with RvD1 in a antecedent abstraction (30), Gas6 bargain the abundances of p-CaMKII, p-p38, and p-MK2 in accolade siRNA–treated macrophages, and Gas6 bargain the affluence of p–Ser271–5-LOX (Fig. 1A). The bands agnate to the absolute and phosphorylated forms of MK2 and 5-LOX were at the actual atomic accumulation and were absolute by the affirmation of their abridgement in acuteness in siMK2- and siALOX5-treated cells, appropriately (fig. S1A). These furnishings of Gas6 were not apparent in MerTK-silenced macrophages (Fig. 1A), suggesting that Gas6 suppressed the phosphorylation of these kinases through its aftereffect on MerTK. Note that alike beneath ascendancy conditions, that is, with beef incubated with conditioned average from mock-transfected HEK 293 cells, basal p-CaMKII affluence in MERTK siRNA–treated beef was abundantly added compared to that in accolade siRNA–treated macrophages. This was acceptable due to a low bulk of Gas6 in the ascendancy medium, because back beef were incubated in phosphate-buffered acrid (PBS) instead of ascendancy medium, basal p-CaMKII affluence was not afflicted by MERTK siRNA (fig. S1B). Protein S, accession ligand for TAM receptors that activates MerTK, but not Axl (31, 32), additionally suppressed the CaMKII–p38–MK2–5-LOX avalanche (fig. S1C).
(A to C) Beastly monocyte–derived macrophages were transfected with either accolade siRNA or MERTK-specific siRNA. Afterwards 72 hours, the beef were incubated for 7 hours with ascendancy average (Con) or 10 nM Gas6-conditioned medium. (A) Left: The macrophages were lysed and yzed by Western blotting with antibodies specific for phosphorylated (p-) and absolute MerTK, CaMKII, p38, MK2, and 5-LOX. β-Actin served as the loading control. Blots appearance samples from three altered donors. Right: Bar graphs appearance the arrangement of the amounts of phosphorylated protein to absolute protein, which was quantified by densitometry with ImageJ software. Abstracts are agency ± SEM of three altered donors. *P < 0.05, **P < 0.01, and ***P < 0.001 against ascendancy average by one-way appraisal of about-face (ANOVA) with column hoc t tests for accumulation comparisons; n.s., not significant. (B) Average from the adumbrated macrophages was assayed by ELISA to ascertain iLXA4 and iRvD1. (C) During the aftermost hour of Gas6 treatment, macrophages were incubated with or afterwards 10 μM AA to abet leukotriene production. The bulk of iLTB4 in the adeptness average was again yzed by ELISA. Abstracts in (B) and (C) are agency ± SEM of three to six donors. **P < 0.01 and ***P < 0.001 against ascendancy average by one-way ANOVA with column hoc t tests for accumulation comparisons.
Consistent with our antecedent abstracts (14, 30), Gas6 bargain the arrangement of p–Ser271–5-LOX to absolute 5-LOX in a MerTK-dependent address (Fig. 1A). Gas6 additionally added the amounts of immunoreactive lipoxin A4 (iLXA4) and resolvin D1 (iRvD1), as bent by enzyme-linked immunosorbent appraisal (ELISA) measurements, in accolade siRNA–treated macrophages but not in siMERTK-treated macrophages (Fig. 1B). Furthermore, as predicted by the abatement in p–Ser271–5-LOX abundance, Gas6 decreased the bulk of AA-dependent immunoreactive leukotriene B4 (iLTB4) in a MerTK-dependent address (Fig. 1C). These abstracts advance that Gas6, through MerTK, suppresses a kinase avalanche that phosphorylates 5-LOX and accordingly increases lipoxins at the bulk of leukotrienes in beastly macrophages.
With account to how Gas6-MerTK signaling adeptness aish CaMKII activity, we activated whether the alleyway bargain the cytosolic absorption of Ca2 , which would, in turn, abate CaMKII action (33–35). Constant with this possibility, we begin that Gas6 decreased the fluorescence acuteness of the Ca2 indicator Fluo3-AM (36, 37) in a time-dependent address in accolade siRNA–treated macrophages, but not in siMERTK-treated macrophages (Fig. 2A). These breeze cytometric abstracts were accepted by confocal imaging of the beef (fig. S2A). Furthermore, the cytosolic Ca2 chelator BAPTA-AM mimicked the aftereffect of Gas6 in agreement of suppressing the phosphorylation of CaMKII, p38, MK2, and 5-LOX (fig. S2B). Note that the furnishings of Gas6 and BAPTA-AM were not additive, suggesting that they act through the above mechanism.
(A) Beastly macrophages were transfected with accolade siRNA or MERTK-specific siRNA and were incubated with ascendancy or Gas6-conditioned average for the adumbrated times. The beef were again loaded with the Ca2 delving Fluo3-AM, and the cytosolic Ca2 absorption was bent by breeze cytometry. Abstracts are agency ± SEM of three altered donors. **P < 0.01 against accolade siRNA by unpaired t test. (B) Macrophages transfected with accolade RNA or MERTK siRNA were incubated with ascendancy or Gas6-conditioned average for 7 hours. The about abundances of ATP2A2 mRNA (top) and SERCA2 protein (bottom) were assayed by real-time quantitative polymerase alternation acknowledgment (qPCR) and Western blotting ysis, respectively. (C) Macrophages were pretreated with 2 μM thapsigargin (Thaps) for 5 min and again incubated with ascendancy or Gas6-conditioned average for 7 hours. Left: Beef were lysed and yzed by Western blotting with antibodies specific for phosphorylated (p-) and absolute CaMKII, p38, MK2, and 5-LOX. β-Actin served as the loading control. Blots appearance samples from three altered donors. Right: Bar graphs appearance the ratios of the amounts of phosphorylated protein to absolute protein, which were quantified by densitometry with ImageJ software. Abstracts in the bar graphs in (B) and (C) are agency ± SEM of three altered donors. *P < 0.05, **P < 0.01, and ***P < 0.001 against ascendancy average by one-way ANOVA with column hoc t tests for accumulation comparisons.
We advised the antecedent that Gas6-MerTK signaling suppressed CaMKII action by accretion the affluence of SERCA, which transports cytosolic Ca2 into the endoplasmic cloth (38, 39). Gas6 induced increases in ATP2A2, which is the mRNA that encodes SERCA2, and in SERCA2 protein in beastly macrophages in a MerTK-dependent address (Fig. 2B). The Gla area of Gas6, whose gamma carbons are posttranslationally carboxylated, was adapted for the consecration of ATP2A2 because Gla-deleted Gas6 (Gla-less Gas6) (31) was clumsy to abet an access in ATP2A2 (fig. S3A). Moreover, apoptotic beef added ATP2A2 announcement in macrophages (fig. S3B), as did protein S (fig. S3C), which activates MerTK but not Axl. As added affirmation that Axl does not comedy a abounding role in this pathway, appraisal of macrophages with siAXL did not aish the adeptness of Gas6 to abet ATP2A2 (fig. S3D). To added actuate whether SERCA plays a role in the Gas6-mediated aishment of CaMKII activity, we activated whether thapsigargin, a SERCA inhibitor (40, 41), blocked Gas6-mediated aishment of the kinase pathway. As predicted, thapsigargin aished the Gas6-mediated aishment of the phosphorylation of CaMKII, p38, MK2, and 5-LOX (Fig. 2C). Constant with the proposed arrangement of this pathway, we begin that ATP2A2 was added afterwards 3 hours of Gas6 treatment, admitting p–5-LOX was not decreased until 5 hours afterwards dispatch (fig. S3, E and F). Together, these abstracts abutment the antecedent that Gas6-MerTK signaling suppresses CaMKII phosphorylation and the consecutive afterwards alleyway by inducing ATP2A2 announcement and abbreviation cytosolic Ca2 .
Previous studies accept apparent that Gas6-MerTK signaling activates ERK in assorted corpuscle types, including macrophages (42–45), and that ERK1/2 induces ATP2A2 in abrasion macrophages (46). We accordingly advised whether Gas6-MerTK–induced ATP2A2 was advised through ERK1/2 activation. Gas6, protein S, and apoptotic beef added the affluence of p-ERK in macrophages (Fig. 3A and fig. S4, A and B), and the Gas6-induced access in p-ERK was essentially bargain in MerTK-silenced macrophages compared to that in ascendancy beef (Fig. 3A). ERK activation was upstream of ATP2A2 consecration and the consecutive CaMKII–p38–MK2–5-LOX pathway, because both U0126, an inhibitor of the ERK-activating kinases MEK1/2 (47, 48), and ERK-specific siRNA (siERK) suppressed the Gas6-induced announcement of ATP2A2 mRNA and SERCA2 protein, as able-bodied as the phosphorylation of apparatus of the CaMKII–p38–MK2–5-LOX avalanche (Fig. 3, B and C, and fig. S4, C and D). Constant with these data, U0126 blocked the adeptness of Gas6 to access the affluence of iLXA4 and to abatement that of iLTB4 (Fig. 3D).
(A) Beastly macrophages transfected with accolade siRNA or MERTK-specific siRNA were incubated with ascendancy or Gas6-conditioned average for 30 min, lysed, and again yzed by Western blotting with antibodies specific for MerTK, p-ERK1/2, ERK1/2, and β-actin (loading control). Left: Western blots appearance samples from three altered donors. Right: Densitometric appraisal of the arrangement of the abundances of p-ERK1/2 to absolute ERK1/2. (B) Macrophages were pretreated with agent or 10 μM U0126 for 30 min and again incubated with ascendancy or Gas6-conditioned average for 7 hours. The beef were again yzed by real-time qPCR and Western blotting ysis, respectively, to actuate the about abundances of ATP2A2 mRNA (left) and SERCA2 protein (right). Western blots appearance samples from three altered donors. (C) Macrophages were advised as declared in (B) and again were yzed by Western blotting with antibodies specific for phosphorylated (p-) and absolute CaMKII, p38, MK2, and 5-LOX. β-Actin served as the loading control. Left: Western blots appearance samples from three altered donors. Right: Bar graphs appearance the ratios of the adumbrated phosphorylated protein to absolute protein, which were quantified by densitometry with ImageJ software. (D) Macrophages were advised as declared in (B), afterwards which the adeptness average was subjected to ELISAs to quantify iLXA4 or iLTB4. For the macrophages acclimated for the iLTB4 assay, 10 μM AA was added to the average during the aftermost hour of Gas6 treatment. For all bar graphs, abstracts are agency ± SEM of three altered donors. *P < 0.05, **P < 0.01, and ***P < 0.001 by one-way ANOVA with column hoc t tests for accumulation comparisons.
MerTK signaling in added scenarios is abased on tyrosines in its cytoplasmic appendage (49, 50). We accordingly advised whether specific tyrosines in MerTK were adapted for ERK1/2 activation and the aishment of CaMKII phosphorylation. We took advantage of chimeric constructs in which the cytoplasmic appendage of wild-type (WT) abrasion MerTK, “kinase-deficient” (KD) MerTK, or assorted tyrosine-to-phenylalanine MerTK mutants are alloyed with the extracellular area of CD8 (CDMer) (49, 51). Back HEK 293 beef are transfected with plasmid encoding WT CDMer, MerTK signaling is constitutively active, admitting signaling is not apparent in beef transfected with plasmid encoding CDMer-KD (49, 51). We begin that in WT CDMer–expressing cells, there was a apparent access in ERK1/2 phosphorylation compared with that in mock-transfected or CDMer-KD–expressing beef (Fig. 4A). Furthermore, beef cogent CDMer with mutations at Tyr867 and Tyr924 showed actual little ERK1/2 phosphorylation, admitting ERK1/2 phosphorylation was accustomed in beef cogent CDMer with a alteration in Tyr825 (Fig. 4A). We acclimated breeze cytometry to ascertain corpuscle apparent CD8 and begin agnate announcement of the WT and aberrant receptors (fig. S5).
(A) HEK 293 beef were transfected with assorted CDMer plasmids encoding WT MerTK, the Y825F, Y867F, and Y924F mutants, and the KD mutant. Forty-eight hours later, the beef subjected to the transfection action afterwards plasmids served as ascendancy (“Mock”). The beef were lysed and yzed by Western blotting with antibodies specific for p-ERK1/2, ERK1/2, and β-actin. The bar blueprint shows the ratios of phosphorylated ERK1/2 to absolute ERK1/2, which were quantified by densitometry with ImageJ software. Abstracts are agency ± SEM of three absolute experiments. ***P < 0.001 against WT by one-way ANOVA with column hoc t tests for accumulation comparisons. (B) Cartilage marrow–derived macrophages (BMDMs) from Mertk−/− mice were transduced with pMSCV–human MERTK (WT) or pMSCV–human MERTK Y872F. Seventy-two hours later, the beef were again incubated with ascendancy or Gas6-conditioned average for 30 min (left) or 7 hours (right), which was followed by breeze cytometric altitude of p-ERK1/2 or p-CaMKII, respectively. Abstracts are agency ± SEM of three altered mice. **P < 0.01 and ***P < 0.001 against ascendancy average by one-way ANOVA with column hoc t tests for accumulation comparisons. MFI, beggarly fluorescence intensity.
To appraisal the role of Tyr867, we acclimated retroviruses encoding beastly WT MerTK or Y872F-mutant MerTK (Tyr872 in beastly MerTK is agnate to Tyr867 in abrasion MerTK) to transduce macrophages defective autogenous MerTK (macrophages from Mertk–/– mice). Transduced macrophages cogent WT MerTK recapitulated the Gas6-induced access in p-ERK1/2 affluence and abatement in p-CaMKII abundance, and, constant with the aloft abstracts with CDMer-expressing HEK 293 cells, these furnishings of Gas6 were abrogated in macrophages cogent Y872F-mutant MerTK (Fig. 4B). These changes in the abundances of p-ERK1/2 and p-CaMKII were absolute by breeze cytometry (fig. S6A). Breeze cytometry additionally showed that WT and Y872F MerTK were ogously abounding on the corpuscle surface, advertence agnate transfection efficiencies of the two constructs, and that Gas6 induced the phosphorylation of WT but not Y872F-MerTK (fig. S6B). These data, back advised calm with the abstracts from the abstracts application U0126 and siERK, appearance that the adeptness of Gas6 to actuate the SERCA–CaMKII–5-LOX alleyway depends on ERK1/2 activation through a apparatus acute specific tyrosine residues in the cytoplasmic appendage of MerTK.
To actuate whether the MEK-ERK alleyway was adapted for resolution of deepening in vivo, we angry to zymosan-induced peritonitis, a archetypal in which resolution has been apparent to be abased on MerTK (30). WT mice were injected intraperitoneally with zymosan in the absence or attendance of U0126, and again the cardinal of address neutrophils was abstinent over time to chase the deepening and resolution stages. The cardinal of neutrophils ailing at 12 hours (Tmax) and again beneath in both groups of mice, but the abatement was slower in the U0126-treated mice: the time to a 50% abridgement of the aiguille bulk (T50) was ~21 hours in agent [dimethyl sulfoxide (DMSO)]–treated mice as compared to ~29 hours in the U0126-treated mice, acquiescent resolution intervals (Ri = T50− Tmax) of 9 and 17 hours, appropriately (Fig. 5A). These abstracts are constant with the angle that MEK1/2 inhibition delays deepening resolution.
(A) WT mice were co-injected intraperitoneally with 0.1 mg of zymosan and either U0126 (25 μg/kg) or an according aggregate of DMSO as the agent control. Peritoneal exudates were calm by lavage with 3 ml of algid PBS at the adumbrated times, and leukocytes and address aqueous were afar by centrifugation at 500g for 10 min. Absolute corpuscle cardinal in the address was counted with a hemocytometer, and the allotment of Ly6G neutrophils was bent by breeze cytometry. Neutrophil cardinal was affected as absolute leukocytes × allotment of neutrophils. Resolution intervals (Ri) were affected as ahead declared (30). Abstracts are agency ± SEM of four mice per group. **P < 0.01 and ***P < 0.001 against vehicle-treated mice by unpaired t test. (B to D) WT or Mertk−/− mice were injected intraperitoneally with zymosan, which was followed 64 hours afterwards by intraperitoneal bang with U0126 or DMSO. Afterwards an added 8 hours, peritoneal exudates were collected. (B) Neutrophil cardinal was affected as declared in (A). (C) Peritoneal leukocytes were decrepit with phycoerythrin (PE)–conjugated anti-F4/80 antibody, which was followed by fixation and permeabilization. Permeabilized beef were decrepit with anti–p-CaMKII antibiotic and again an Alexa Fluor 647–conjugated accessory antibody. The MFI of p-CaMKII in the macrophages was quantified by breeze cytometry. (D) Address iLXA4 was assayed by ELISA. For (B) to (D), abstracts are agency ± SEM of four mice per group. *P < 0.05 and ***P < 0.001 against vehicle-treated WT mice by one-way ANOVA with column hoc t tests for accumulation comparisons.
In the aing set of experiments, we injected the mice with U0126 during the aeon back absolute macrophages accrue in the abdominals (64 hours afterwards bang with zymosan) (52, 53) and again conducted our assays 8 hours later. We additionally compared WT and Mertk−/− mice in this agreement to actuate whether MerTK and MEK-ERK inhibition acted in the above alleyway to actuate resolution. As predicted from the beforehand abstracts (Fig. 5A), the cardinal of neutrophils was greater in the U1026-treated WT mice (Fig. 5B), advertence broken resolution (14, 30). As appear ahead (30), accident of MerTK additionally resulted in broken resolution, and the furnishings of U0126 and MerTK abatement were not accretion (Fig. 5B, additional brace of bars). The bulk of p-CaMKII in peritoneal macrophages from zymosan-treated WT mice was additionally added by U0126 and MerTK abatement in a nonadditive address (Fig. 5C), and the bulk of proresolving iLXA4 in these beef was decreased by both U0126 and MerTK deletion, additionally in a nonadditive address (Fig. 5D). These abstracts advance that the MerTK–ERK–CaMKII-lipoxin alleyway functions to dness deepening in vivo (fig. S7).
The abstracts actuality acknowledge a signaling alleyway in beastly macrophages that links MerTK activation with the aishment of CaMKII activity, LXA4 and RvD1 synthesis, and the resolution of inflammation. A key catechism is how MerTK becomes activated in vivo to bedew deepening and advance resolution. Acceptable candidates accommodate apoptotic beef and Gas6, both of which access in affluence with deepening (54, 55). Added MerTK ligands that adeptness be complex accommodate protein S, Tubby, tubby-like protein 1 (Tulp1), and galectin-3 (56). However, the about accession of altered MerTK activators in deepening and resolution charcoal alien and about absolutely varies in altered physiologic and pathophysiologic settings. MerTK signaling additionally suppresses the transcriptional action of NF-κB in acknowledgment to lipopolysaccharide (LPS) (27, 50, 57, 58), suggesting that MerTK both suppresses deepening and enhances resolution through abstracted signaling pathways and effector mechanisms. Furthermore, the NF-κB–suppressing action of MerTK, agnate to its proresolution action declared here, requires the attendance of Tyr867 in its cytoplasmic appendage (50).
A key advocate in the MerTK-induced resolution of deepening is the aishment of CaMKII activity, which is additionally the case with RvD1-mediated resolution in macrophages (14). In this context, a abstraction from our accumulation showed that in fat-fed Ldlr−/− mice, abiogenetic targeting of Camk2g in myeloid cells, which encodes the above isoform of myeloid CaMKII, CaMKIIγ, improves efferocytosis, decreases necrosis, and increases coarse cap array in avant-garde atheroscl lesions, suggesting an bigger resolution acknowledgment (59). Furthermore, myeloid cell–specific abatement of Camk2g led to an access in cell-surface MerTK through a alleyway involving consecration of the Mertk inducer, alarmist X receptor α (LXRα) (59). These allegation are evocative of a absolute acknowledgment aeon in deepening resolution and efferocytosis in which MerTK activation suppresses CaMKII activity, which again leads to added MerTK abundance. The improvements in atheroscl applique endpoints apparent in mice with myeloid cell–specific CaMKII abatement were actual agnate to those apparent in Ldlr−/− mice with genetically added MerTK activity, and in that case, we showed anon that the arrangement of SPMs to leukotrienes in atheroscl lesions was added (26). In accession book accordant to resolution, mice defective CaMKII in cardiomyocytes display a blunted anarchic acknowledgment afterwards myocardial infarction (60). As with genetically added MerTK activity, abatement of CaMKII may additionally anon aish inflammation. For example, the anarchic acknowledgment in macrophages advised with LPS and added Toll-like receptor activators in vitro has been associated with CaMKII activation (61–63).
Activated receptor tyrosine kinases can recruit advance agency receptor–binding protein 2 (Grb2) to their phosphorylated tyrosine residues, arch to the activation of a Ras–Raf–MEK–ERK1/2 avalanche (64). Tyr872 in beastly MerTK (which is agnate to Tyr867 in murine MerTK) binds to Grb2 and initiates corpuscle adaptation signaling (51, 65). Constant with this finding, we begin that Tyr872 was adapted for Gas6-induced ERK activation and aishment of CaMKII activity. Note that ERK can actuate both anarchic and anti-inflammatory responses (66–68). With attention to our findings, ERK cooperates with SPMs to advance resolution. A antecedent abstraction showed that, inhibition of ERK by a MEK1 inhibitor aishes RvE1-mediated phagocytosis of zymosan A by beastly macrophages (69). As accession example, annexin A1, a proresolving mediator, activates ERK and inhibits the alternation of neutrophils with the endothelium (70). Furthermore, ERK1/2 signaling is activated in macrophages in which apoptotic beef were accustomed by the efferocytosis receptor LDL (low-density lipoprotein) receptor–related protein 1 (LRP1), and ERK activation again facilitates internalization of the apoptotic beef by the macrophages (71). Thus, it is accessible that LRP1-mediated efferocytosis additionally activates the resolution alleyway declared actuality and that ERK activation in MerTK-activated macrophages contributes to the internalization of apoptotic beef in accession to acceptable resolution. Finally, abrasion macrophages that are aggressive to the accomplishments of insulin, which action in the ambience of blazon 2 diabetes, appearance ERK inhibition-mediated aishment of ATP2A2 mRNA and SERCA2 protein and burning of endoplasmic cloth Ca2 , suggesting that ERK prevents SERCA-mediated Ca2 absolution into the cytoplasm by inducing in this ambience (46). These findings, back advised with our allegation and those in the aloft CaMKII-atherosclerosis study, advance a accessible apparatus bond insulin attrition and blazon 2 diabetes to accelerated atheroscl vascular disease.
In summary, we accept elucidated a ahead uncharacterized MerTK signaling alleyway in macrophages that leads to an added resolution response. In appearance of the role of broken resolution in abounding ytical abiding anarchic diseases, the ameliorative abeyant of these allegation warrants consideration. On the one hand, absolute administering of absolute mediators is ability all-encompassing preclinical testing for assorted anarchic diseases, with aboriginal beastly trials in advance (72). However, the achievability of aesthetic a accustomed alleyway of absolute advocate amalgam is appealing. The challenges of leveraging the alleyway declared actuality for that purpose accommodate anecdotic specific MerTK activators that do not accept non–MerTK-mediated adverse furnishings (56), such as those that accept been active in oncogenesis (73). In-depth abstraction of MerTK signaling pathways in altered in vivo settings will be bare to actuate whether and how these challenges can be affected (74).
To accomplish beastly macrophages, we abandoned monocytes from the buffy coats of de-identified advantageous volunteers (New York Blood Center) as ahead declared (30). Briefly, buffy coats were acclaim layered assimilate Histopaque band-aid (Sigma-Aldrich) as 1:1 arrangement (v/v) and centrifuged at 1500g for 25 min. Leukocytes were removed from the average layer, done with RPMI, and again centrifuged at 1500g for 5 min. This ablution footfall was again once, and again the corpuscle pellet was abeyant in RPMI and argent into 12-well plates. Afterwards 3 to 4 hours, back monocytes were adherent, the average was exchanged for RPMI absolute 10% (v/v) fetal bovine serum (FBS), 1% penicillin-streptomycin, and recombinant beastly granulocyte-macrophage colony-stimulating agency (10 ng/ml; PeproTech), and the beef were incubated for 7 to 10 canicule to acquiesce macrophage differentiation. To accomplish abrasion BMDMs, cartilage bottom beef were able in Dulbecco’s adapted Eagle’s average (DMEM) absolute 10% (v/v) FBS, 1% penicillin-streptomycin, and macrophage colony-stimulating factor, as declared ahead (14, 30).
Human macrophages were incubated in average absolute 50 nM ON-TARGETplus Beastly MERTK siRNA, MK2 siRNA, ALOX5 siRNA, ERK siRNA, or accolade siRNA (Dharmacon) and Lipofectamine RNAiMAX (Thermo Fisher Scientific). Afterwards 72 hours, the abstracts were conducted as adumbrated in the amount legends.
Conditioned average absolute γ-carboxylated Gas6 was harvested from beastly Gas6-expressing HEK 293-6E beef incubated with vitamin K (2 μg/ml), as declared ahead (30, 32). The absorption of Gas6 in the average was 250 nM as bent by comparing the Gas6 arresting from a Western blot, quantified by densitometry, with that of a accepted ambit generated through Western blotting and altitude of assorted concentrations of bartering recombinant beastly Gas6 (R&D Systems). Macrophages were done with PBS three times, preincubated for 1 hour in serum-free DMEM, and again incubated in serum-free DMEM with a 1:25 concoction of conditioned medium, agnate to 10 nM γ-carboxylated Gas6, or an according aggregate of conditioned average from nontransfected HEK 293 cells, which is referred to in the abstracts as “control” (Con) at the adumbrated times. Alternatively, macrophages were advised with 100 nM protein S, which was antiseptic from beastly claret (Haematologic Technologies).
Cell extracts were bound on 4 to 20% acclivity SDS-polyacrylamide gels and transferred to 0.45-μm nitrocellulose membranes. The membranes were blocked in tris-buffered saline/0.1% Tween 20 (TBST) absolute 5% (w/v) nonfat milk at allowance temperature for 1 hour and again incubated with the primary antibiotic in TBST absolute 5% (w/v) nonfat milk or 5% (w/v) BSA (bovine serum albumin) at 4°C overnight. The membranes were again incubated with the adapted accessory antibiotic accompanying to horseradish peroxidase (HRP), and proteins were detected with the ECL SuperSignal West Pico Chemiluminescent Substrate kit (Thermo Fisher Scientific). Antibodies acclimated for the Western blotting were as follows: anti–p-CaMKII (Thr286) (Novus); anti-CaMKII (Santa Cruz Biotechnology); anti-SERCA2, anti–p-ERK1/2 (Thr202/Tyr204), anti–p–5-LOX (Ser271), anti–5-LOX, anti–p-MK2 (Thr334), anti-MK2, anti–p-p38 MAPK (Thr180/Tyr182), anti–p38 MAPK, and anti–HRP–β-actin (Cell Signaling Technology); anti–p-MerTK (PMKT-140AP, FabGennix); and anti-MerTK (ab52968, Abcam).
Human macrophages in 12-well plates were incubated for 7 hours at 37°C with agent or 10 nM γ-carboxylated Gas6. The corpuscle adeptness average was again harvested for appraisal by (i) the Neogen LXA4 ELISA kit, which has the afterward cross-reactivities according to the manufacturer: 15-epi-LXA4 (24%), 5(S),6(R)-DiHETE (5%), LXB4 (1%), and 15-HETE (0.1%); (ii) the Cayman LTB4 ELISA kit, which has cross-reactivities to 5,6-DiHETE (0.07%), 5(R)-HETE (3.7%), 15(R)-HETE (0.98%), 15(S)-HETE (0.4%), 5(S)-HETE (6.6%), 20-hydroxy LTB4 (2.7%), 6-trans-12-epi LTB4 (0.31%), and 6-trans LTB4 (0.11%); or (iii) the Cayman RvD1 ELISA kit, which has cross-reactivities to 5(S),6(R)-LXA4 (20%), 17(R)-RvD1 (4.2%), and 10(S),17(S)-DiHDoHE (0.7%). We accredit to these lipid mediators as iLXA4, iLTB4, and iRvD1, respectively, area “i” stands for “immunoreactive.” The abstracts are appear as bend change in affluence in the beginning accumulation about to that in the ascendancy group, which was set at 1.0. Agnate yses were conducted on peritoneal exudates for the zymosan experiments. In all cases, 50 μl of sample was assayed as per the manufacturer’s instructions.
Human macrophages were loaded with 1.25 μm of Fluo3-AM (Thermo Fisher Scientific) for 30 min at allowance temperature in loading absorber absolute 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 20 mM Hepes, 10 mM glucose, 1× PowerLoad (Thermo Fisher Scientific) to solubilize the Fluo3-AM dye, and 2.5 mM probenecid (Thermo Fisher Scientific) for dye retention. Fluo3-AM was again removed, and the beef were chased in loading absorber afterwards PowerLoad and probenecid for 30 min at allowance temperature. Cytosolic Ca2 was monitored by breeze cytometry (FACSCanto II) or with a Nikon A1 confocal microscope.
Retroviral announcement vectors pLXSN cogent a chimeric receptor (CDMer) generated from the extracellular and transmembrane domains of beastly CD8 (amino acids 1 to 209) and the intracellular arena (amino acids 521 to 994) of WT MerTK, K614M KD MerTK, Y825F MerTK, Y867F MerTK, or Y924F MerTK were generated and acclimated to transfect HEK 293T beef as declared ahead (49, 50).
Human MERTK (NM_006343.2) was cloned into pMSCV-puro retroviral agent (Addgene) at the Xho I and Eco RI brake sites (Genewiz). Tyr872 of beastly MERTK was mutated to phenylalanine application the PCR-based QuikChange mutagenesis arrangement (Stratagene). BOSC23 beef (5 × 106 cells) were transfected with 1 μg of pMSCV-WT Mertk or pMSCV-Y872F MERTK calm with 1 μg of pCL-Eco (Addgene) and 2 μg of pMD2.G (Addgene) application 16 ml of LipoD293 transfection reagent (SignaGen). Conditioned average was calm 48 hours afterwards transfection and filtered through 0.45-μm filters. The average was again acclimated to transduce BMDMs from Mertk−/− mice.
Eight- to 10-week-old C57BL/6J mice were injected intraperitoneally with 0.1 mg of zymosan A (Sigma-Aldrich) per mouse. Peritoneal exudates were calm by lavage with 3 ml of algid PBS afterwards 72 hours. Peritoneal leukocytes and address fluids were afar by centrifugation at 500g for 10 min. Mice were about assigned to appraisal groups. All procedures were conducted in accordance with the guidelines for beastly affliction of the Columbia University Institutional Beastly Affliction and Use Committee.
For the zymosan-induced peritonitis experiments, peritoneal address beef harvested at the times adumbrated in the amount fable were done with fluorescence-activated corpuscle allocation (FACS) staining absorber [PBS absolute 3% (v/v) FBS]. The beef were incubated for 5 min at 4°C with Fc Block (BD Biosciences) and again labeled with Pacific Blue–Ly6G (clone 1A8, eBioscience) to ascertain polymorphonuclear neutrophils. To ascertain p-CaMKII in address macrophages, beef were decrepit with PE-F4/80 (clone BM8, eBioscience) for 30 min at 4°C to stain macrophages, which was followed by fixation and permeabilization of the cells. Permeabilized beef were again incubated with aerial anti–p-CaMKII for 1 hour at 4°C and again with Alexa Fluor 647–conjugated dupe anti-rabbit accessory antibiotic for 30 min at 4°C. For the breeze cytometric appraisal of p-ERK and p-CaMKII in WT MerTK–expressing against Y872F MerTK–expressing Mertk−/− macrophages, the beef were aboriginal incubated with allophycocyanin (APC)–MerTK (clone #125518, R&D Systems) to characterization transduced macrophages. Afterwards fixation and permeabilization, the beef were incubated with aerial anti–p-ERK or anti–p-CaMKII antibodies, which was followed by evolution with PE-conjugated anti-rabbit accessory antibody. The beef were abeyant in FACS absorber and yzed for the MFI of p-ERK and p-CaMKII in APC-MerTK beef gated application a FACSCanto II (BD Biosciences) breeze cytometer and FlowJo software.
Jurkat beef were apparent to ultraviolet ablaze at 254 nm for 5 min application a lamp from Ultra-Violet Products Ltd. and again incubated in a 37°C incubator with 5% CO2 for 3 hours to abet apoptosis as ahead declared (75). Apoptotic Jurkat beef (ACs) were added to beastly macrophages in the attendance of FBS-containing average for the times adumbrated in the amount legend. ACs were again removed by rinsing bristles times with algid PBS, and the abundances of ATP2A2 mRNA or p-ERK and absolute ERK were assayed as adumbrated in the amount legends.
All after-effects are apparent as agency ± SEM. Two-tailed P ethics were affected application Student’s t appraisal for aloof two groups. With added than two groups, one-way ANOVA with column hoc t tests was acclimated for accumulation comparisons. One-way ANOVA was additionally acclimated with two factors, for example, genotype and treatment, to acquiesce for interaction. For example, in Fig. 5, U0126 furnishings could be altered amid WT and Mertk−/−.
Fig. S1. Validation of the MK2 and 5-LOX antibodies with siRNAs; MerTK abatement does not adapt CaMKII action in the absence of Gas6; and protein S can aish the CaMKII pathway.
Fig. S2. Gas6 reduces cytosolic Ca2 in a MerTK-dependent manner, and BAPTA-AM suppresses CaMKII–p38–MK2–5-LOX signaling.
Fig. S3. Added studies accompanying to MerTK ligands and affirmation that Axl does not arbitrate Gas6-induced ATP2A2 expression.
Fig. S4. Protein S and apoptotic beef actuate ERK1/2 in macrophages, and ERK1/2 is adapted for Gas6-mediated aishment of CaMKII–p38–MK2–5-LOX signaling.
Fig. S5. Detection of the corpuscle apparent announcement of CDMer proteins in transfected HEK 293 cells.
Fig. S6. Tyr872 in the cytoplasmic appendage of beastly MerTK is adapted for the activation of ERK1/2 and CaMKII.
Fig. S7. Arbitrary arrangement of MerTK-mediated resolution signaling.
Funding: This assignment was supported, in part, by an American Heart Association Postdoctoral Fellowship admission (to B.C.); NIH admission 1K99DK115778 (to B.C.); NIH admission CA165077 and a admission from the New Jersey Health Foundation (to R.B.B.); and NIH grants HL132412, HL075662, and HL127464 (to I.T.). The fluorescence microscopy abstracts acclimated the Confocal and Specialized Microscopy Core at Columbia University’s Irving Cancer Research Center, and the breeze cytometry yses acclimated the Columbia Center for Translational Immunology/Diabetes and Endocrinology Research Center Breeze Core facility, funded, in part, by NIH/National Institute of Diabetes and Digestive and Kidney Diseases Center Admission 5P30DK063608. Author contributions: B.C. and I.T. accomplished the study. R.B.B. provided ytical ascribe in the activity design. B.C., C.K., and A.C.D. conducted the experiments. All authors contributed to the abstracts ysis, and R.R. was the statistical adviser for the study. B.C. and I.T. wrote the antecedent arrangement draft, which was again advised and edited by the blow of the authors. Aggressive interests: The authors acknowledge that they accept no aggressive interests. Abstracts and abstracts availability: All abstracts bare to appraise the abstracts of this abstraction are accessible in the cardboard or the Supplementary Materials.
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