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Stimulation of Vibratory Urticaria-Associated Adhesion-GPCR, EMR2/ADGRE2, Triggers the NLRP3 Inflammasome Activation Signal in Human Monocytes

EMR2/ADGRE2 is an adhesion G protein-coupled receptor differentially expressed by human myeloid cells. It modulates diverse cellular functions of innate immune cells and a missense EMR2 variant is directly responsible for vibratory urticaria. Recently, EMR2 was found to activate NLRP3 inflammasome in monocytes via interaction with FHR1, a regulatory protein of complement Factor H. However, the functional involvement of EMR2 activation and its signaling mechanisms in eliciting NLRP3 inflammasome activation remain elusive. In this study, we show that EMR2-mediated signaling plays a critical role in triggering the activation (2nd) signal for the NLRP3 inflammasome in both THP-1 monocytic cell line and primary monocytes. Stimulation of EMR2 by its agonistic 2A1 monoclonal antibody elicits a Gα16-dependent PLC-β activation pathway, inducing the activity of downstream Akt, MAPK, NF-κB, and Ca2+ mobilization, eventually leading to K+ efflux. These results identify EMR2 and its associated signaling intermediates as potential intervention targets of NLRP3 inflammasome activation in inflammatory disorders.


INTRODUCTION
Proteolytic conversion of procaspase-1 to active caspase-1 and the subsequent processing and secretion of inflammatory IL-1b and IL-18 are the hallmarks of inflammasome activation (1,2). Although it is a critical immune response to microbial infection and tissue injury, dysregulated inflammasome activities are often linked to pathological manifestations such as autoinflammatory, autoimmune, and metabolic disorders as well as cancer (3)(4)(5). The NLRP3 inflammasome is a large assembly of multimeric NLRP3, ASC and procaspase-1, and represents the principal inflammasome activated in innate immune cells through recognition of pathogen-associated molecular patterns (PAMPs) and/or damage-associated molecular patterns (DAMPs) (6,7). The canonical NLRP3 inflammasome activation pathway is normally mediated by a two-signal mechanism; the priming (1 st ) signal is induced by PAMPs and the activation (2 nd ) signal elicited by diverse cellular stressors or DAMPs such as extracellular ATP, poreforming toxins and particulates (6,8). While the priming signal is mostly transmitted by pattern recognition receptors (PRRs), the activation signal can be triggered by an increasing number of different cell surface proteins including G protein-coupled receptors (GPCRs) (8,9). As GPCRs are extensively-proven drug targets, identification and characterization of novel GPCRs involved in NLRP3 inflammasome activation not only can advance our understanding of this important immune reaction, but also be exploited for potential therapeutics of inflammasomeassociated disorders (10).
EMR2/ADGRE2 is a human-specific paralogue of the wellknown mouse tissue macrophage (Mj)-specific F4/80 antigen (Ag) (11). Both EMR2 and F4/80 belong to the ADGRE/EGF-TM7 subfamily of adhesion GPCRs (aGPCRs) that is distinguished by multiple extracellular epidermal growth factor (EGF)-like domains (12)(13)(14). Unlike F4/80, however, EMR2 is more widely expressed and differentially regulated in cells of the myeloid lineage including monocytes, Mj, neutrophils (Nj), and myeloid dendritic cells (DCs) (13,15). EMR2 expression is up-regulated restrictedly within inflamed tissues and its levels on Nj are positively correlated with the severity and overall mortality of patients of systemic inflammatory response syndrome (SIRS) and liver cirrhosis (16,17). These findings indicated an immune regulatory function for EMR2. Indeed, EMR2 activation has been shown to potentiate adhesion, migration and anti-microbial activities of Nj in response to diverse inflammatory stimuli (18)(19)(20). A missense EMR2-pC492Y variant was identified recently as the cause of familial vibratory urticaria (VU), a rare autosomal dominant dermal allergic disorder resulting from vibration-induced mast cell activation (21).
We have previously identified dermatan sulphate (DS) as an endogenous glycosaminoglycan ligand of EMR2 (22). DS-EMR2 interaction plays a potential role in recruiting monocytes to the inflamed synovium in rheumatoid arthritis (RA) (23). Interaction of the VU-inducing EMR2-pC492Y variant with DS or a specific 2A1 monoclonal antibody (mAb) sensitizes mast cells for hyperdegranulation upon vibratory stimulation, indicating a mechanosensing role for EMR2 (21). Our earlier results revealed that 2A1-induced EMR2 activation in monocytic cells lead to Mjlike phenotypes and enhanced production of IL-8 and TNF via a Ga 16 -dependent pathway activating the downstream effectors, including phospholipase C (PLC)-b, PI3K, Akt, MAPK, and NF-kB (24). Interestingly, 2A1-induced EMR2 signaling occurred only when the mAb was immobilized on the culture plates (24). In a recent study, complement Factor H-related protein 1 (FHR1) was shown to function both as a sensor of necrotic cells and a specific serum ligand of EMR2 (25). Consequently, FHR1 was shown to mark necrotic regions of vasculopathies such as anti-neutrophil cytoplasmic antibody-associated vasculitis (AAV) and atherosclerosis, and concomitantly triggered EMR2-mediated NLRP3 inflammasome activation in monocytes via a Gbg-dependent PLC signaling pathway. Peculiarly, the FHR1-induced EMR2 activation was induced only in the presence of normal human serum (NHS) and immobilized FHR1 (25). Taken together, EMR2 is a human myeloid-restricted aGPCR whose activation and signaling is involved in distinct innate immune functions including NLRP3 inflammasome activation.
In this study, we show that 2A1-induced EMR2 signaling plays a critical role in triggering the NLRP3 inflammasome activation (2 nd ) signal, due to the intracellular K + efflux evoked via a Ga 16dependent PLC-b activation and Ca 2+ mobilization. EMR2 and its downstream signaling effector molecules hence represent novel GPCR-associated targets for intervention of NLRP3 inflammasome activation in relevant inflammatory disorders.

Cytokine ELISA Assay
Unless specified otherwise, cells (2 × 10 6 cells/well) were seeded into 6-well plates pre-coated with or without 2A1 mAb in the absence or presence of various PAMPs, then incubated at 37°C for 24 h. Conditioned medium was collected by centrifugation at 1000x g for 5 min at 4°C and transferred into new 1.5 ml eppendorf tubes. The levels of human IL-8, IL-1b, and IL-18 were measured by DuoSet ® ELISA Development Systems (R&D System) according to the protocols suggested by the manufacturer.

Western Blot Analysis
Cell lysates for Western blot analysis were collected at specific time points as indicated. In brief, cells were harvested by centrifugation at 1500 rpm for 5 min at 4°C, washed once with ice-cold 1× HBSS, and lysed in 100 ml ice-cold modified cell lysis buffer as described previously (24). Proteins were quantified using Bicinchoninic acid (BCA) protein assay kit (PIERCE, Rockford, USA). Proteins were denatured and separated by electrophoresis in SDS-polyacrylamide gels, and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, MA, USA). The blotted PVDF membranes were incubated in blocking buffer (5% of BSA in washing buffer) for 1 h with agitation, followed by incubation with the indicated 1 st Ab (2-4 mg/ml in blocking buffer) for 1 h. The membranes were washed extensively and then incubated with appropriate horseradish peroxidase (HRP)-conjugated 2 nd Ab (1:2000-1:5000 in blocking buffer). Following final extensive washes, bound 2 nd Ab was detected by chemiluminescence (ECL, Amersham Life Science Ltd or SuperSignal West Pico Plus, Pierce).

Statistical Analysis
Quantitative analysis was based on results of at least three independent experiments unless indicated otherwise. Data were analyzed using one-way ANOVA analysis by Prism 5 software (GraphPad Software Inc., La Jolla, California, USA) and shown as means ± standard error of the mean (SEM). The statistical significance of p value was set at *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Ligation and Activation of EMR2 Receptor in Human Monocytic Cells Enhance IL-1b and IL-18 Production in the Presence of PAMPs
To investigate the role of EMR2-induced signaling in inflammasome activation, THP-1 monocytic cells were cultured on plates coated with EMR2-specific 2A1 mAb in the absence or presence of various PAMPs. Low but significant levels of IL-1b were noted when cells were stimulated with 2A1 or LPS individually. However, much higher IL-1b levels were detected when cells were treated simultaneously with 2A1 and LPS, indicating a synergistic effect ( Figure 1A). By contrast, neither the isotype control nor the soluble 2A1 mAb had any effect on IL-1b production (Supplementary Figure 1A). Importantly, the specificity of 2A1induced EMR2 activation in up-regulating IL-1b was verified in cells transfected with EMR2-specific siRNAs ( Figure 1B and Supplementary Figure 1B). These results were in line with our previous data and hence all following experiments were performed using immobilized 2A1 mAb (24). Similar 2A1induced IL-1b up-regulation was obtained in cells treated with conventional or ultrapure LPS, and this effect was both concentration-and time-dependent; ranging from 5-10 mg/ml of 2A1 mAb and starting as early as 4 h of incubation (Figures 1A, C and Supplementary Figure 1C). In parallel, significantly higher IL-18 levels were produced by cells co-stimulated with 2A1 plus LPS or 2A1 plus Pam3CSK4 (TLR2/TLR1 ligand) than those treated with the stimulus alone ( Figure 1D). Finally, enhanced levels of IL-1b were also identified in cells co-stimulated with 2A1 and various TLR and NLR ligands including Pam3CSK4, FLA-ST (TLR5 ligand) and MDP (NOD2 ligand)( Figure 1E). These results suggest that EMR2 activation can collaborate with the signaling of diverse PRRs to up-regulate IL-1b and IL-18 production in monocytic cells.
Along with the time-dependent IL-1b up-regulation, increased levels of pro-IL-1b and active caspase-1 were detected in the cell lysate and conditioned medium, respectively, of cells co-treated with LPS and 2A1 than those treated individually with 2A1 or LPS (Figures 2A, B). Conversely, EMR2-induced IL-8 levels remained unchanged in the presence of LPS, indicating a specific synergistic effect of EMR2 activation on IL-1b up-regulation ( Figure 2A). Critically, EMR2-induced IL-1b and IL-18 up-regulation was markedly diminished in cells pre-treated with an irreversible caspase-1 inhibitor, z-YVAD-fmk, confirming the essential role of active caspase-1 in EMR2-promoted IL-1b and IL-18 production ( Figures 2C, D). Taken together, activation of EMR2 in THP-1 cells by immobilized, but not soluble, 2A1 mAb significantly up-regulates IL-1b and IL-18 production in the presence of PAMPs, most likely due to inflammasome activation.

EMR2 Stimulation Activates the NLRP3 Inflammasome
The NLRP3 inflammasome is the most common inflammasome activated by diverse PAMPs in monocytes and was reported to be elicited by FHR1-EMR2 interaction (25). We hence examined the role of 2A1-induced EMR2 ligation in NLRP3 inflammasome activation by first performing gene-specific siRNA knock down targeting ASC and NLRP3. As shown, up-regulated IL-1b production induced by the combined LPS and 2A1 treatment was greatly attenuated in THP-1 cells transfected with the ASC- or NLRP3-specific siRNAs, but not the scrambled control siRNA. Concomitantly, reduced levels of active caspase-1 were found in ASC-and NLRP3-knock down cells (Figures 3A, B).
Thereafter, we employed commercially available ASCdeficient (def-ASC) and NLRP3-deficient (def-NLRP3) THP-1 cell lines to clarify the role of EMR2-induced signaling in activating the NLRP3 inflammasome. Comparable EMR2 expression levels were detected in all three THP-1 cell lines, suggesting no direct roles for ASC and NLRP3 in regulating EMR2 expression (Supplementary Figure 1D). As expected, the enhanced IL-1b levels induced by 2A1 plus LPS in parental THP-1 cells were greatly diminished in def-ASC and def-NLRP3 cells ( Figure 3C). Likewise, notably reduced levels of IL-1b and active caspase-1 were detected in the conditioned medium of def-ASC and def-NLRP3 cells ( Figure 3D). Conversely, comparable IL-8 levels were induced in all three cell lines treated with 2A1, LPS, or 2A1 plus LPS, again supporting the specific role of ASC and NLRP3 in EMR2-promoted IL-1b production ( Figure 3C). Collectively, these results establish a role for EMR2-mediated signaling in promoting NLRP3 inflammasome activation.

EMR2 Ligation Triggers the Activation Signal for NLRP3 Inflammasome Activation
In accordance with the canonical two-signal activation mechanism of NLRP3 inflammasome (8,9), our results suggested that 2A1activated EMR2 most likely elicited the activation (2 nd ) signal.
To validate this, we incubated THP-1 cells with 2A1 in the absence or presence of exogenous ATP or monosodium urate (MSU) crystal, two typical triggers of the activation signal. As reported elsewhere, ATP and MSU treatments lead to significant IL-1b upregulation in the presence, but not in the absence, of LPS. However, no apparent IL-1b enhancement was detected in cells co-stimulated with 2A1 plus ATP or MSU versus cells treated with 2A1 alone ( Figure 4A). Additionally, ASC oligomerization, a signature feature of NLRP3 inflammasome assembly and activation, was more readily identified in cells co-treated with LPS and 2A1 than those treated singly with LPS or 2A1 ( Figure 4B,  Supplementary Figure 2). These results indicate strongly that 2A1induced EMR2 activation predominantly provides the activation signal for NLRP3 inflammasome activation.
Unlike murine myeloid cells, human primary monocytes promptly secrete mature IL-1b in response to the PAMPtriggered priming signal alone. This is mainly due to the heightened responsiveness of human primary monocytes, leading to the release of endogenous ATP or activation of the "alternative" inflammasome activation pathway following PAMP stimulation (27)(28)(29)(30). To attest to the role of EMR2 ligation in inducing the NLRP3 inflammasome activation signal, primary CD14 + monocytes were isolated and incubated with 2A1 in the absence or presence of LPS. In line with previous reports, significant IL-1b production was induced in cells receiving LPS treatment alone. Importantly, cells treated with LPS plus 2A1 produced markedly higher IL-1b levels, while those stimulated with 2A1 alone generated only basal IL-1b levels as did control  Figures 5A-D). Again, the enhanced IL-1b induced by LPS or LPS plus 2A1 in primary monocytes was significantly attenuated in the presence of a selective inhibitor of caspase-1, Ac-YVAD-CHO ( Figures 5C, D).
During the course of this study, Irmscher et al. identified the complement system protein FHR1 as an EMR2-specific ligand and showed that FHR1-EMR2 interaction triggered NLRP3 inflammasome activation in primary monocytes (25). Intriguingly, the FHR1-induced NLRP3 inflammasome activation took place only in the presence of normal human serum (NHS) and when FHR1 was immobilized (25). These findings are somewhat similar to ours and prompted us to investigate the effect of NHS on 2A1-induced EMR2 activation. Interestingly, combined treatment of NHS and 2A1 generated little effect on IL-1b production in primary monocytes in comparison to cells treated with NHS or 2A1 alone. Nevertheless, NHS was efficient in up-regulating IL-1b production by monocytes treated with LPS or LPS plus 2A1 ( Figure 5E). Likewise, addition of NHS, ATP, or both in the monocyte culture increased IL-1b levels significantly only in the presence of LPS ( Figure 5F). Similar results were also observed in THP-1 cells subjected to the same culture conditions (Supplementary Figure 3). Hence, unlike in the case of FHR1, NHS is not required for 2A1-induced EMR2 activation. We conclude that the major role of 2A1-elicited EMR2 signaling is to trigger the activation (2 nd ) signal for NLRP3 inflammasome activation.

EMR2-Induced NLRP3 Inflammasome Activation Signal Is Mediated via the PLCb/Akt/Ca 2+ /MAPK/NF-kB Axes Downstream of Ga 16
Our previous studies have established that 2A1-elicited EMR2 signaling activated the Ga 16 /PLC-b/PI3K/Akt/MAPK/NF-kB axes (24). On the other hand, FHR1-induced EMR2 activation involved a Gbg-dependent PLC-sensitive pathway (25). Ga 16 belongs to the Ga q subfamily that is known to activate PLC-b isoforms, leading to the generation of diacylglycerol (DAG) and inositol triphosphate (IP 3 ), which then triggered MAPK and NF-kB activation as well as intracellular calcium ion (Ca 2+ ) mobilization, respectively (31). Similarly, the Gbg subunits are also able to signal independently via PLC-b isoforms when dissociated from the active Ga subunit (32). To dissect the 2A1-induced EMR2 signaling cascades that triggered the NLRP3 inflammasome activation signal, we performed biochemical analyses using gene-specific siRNAs and diverse signaling inhibitors in THP-1 cells and monocytes. As expected, 2A1-enhanced IL-1b levels were reduced in THP-1 cells transfected with Ga 16 -specific siRNAs but not scrambled siRNA controls (Supplementary Figure 4A). Intriguingly, no significant effects on IL-1b production were observed by Gbg inhibition both in THP-1 cells and monocytes, whereas the use of PLC inhibitor (U73122) and cell-permeable Ca 2+ chelator (BAPTA-AM) attenuated 2A1-enhanced IL-1b release markedly (Figures 6A, B). In parallel, 2A1-induced IL-1b up-regulation was diminished following the blocking of cellular K + efflux in THP-1 cells and monocytes (Figures 6C,  D). These results indicate that 2A1-induced EMR2 activation in monocytes stimulates a Ga 16 -dependent (but Gbg-independent) Ca 2+ mobilization signaling activity leading to intracellular K + efflux, which is generally considered a common trigger of the NLRP3 inflammasome activation signal (confirmed in Supplementary Figure 4B).
In line with our earlier findings, western blot analyses revealed time-dependent phosphorylation and/or activation of Akt, ERK, and IkB following 2A1-induced EMR2 signaling (24).
Critically, these signaling activities were further enhanced in cells co-treated with 2A1 and LPS ( Figure 7A and Supplementary Figure 5A). Concomitantly, EMR2-induced IL-1b up-regulation was mitigated significantly in cells pre-treated with selective inhibitors of ERK (U0126), JNK (SP600125), and IkB kinase (IKK)(Bay 11-7082), but not the p38 inhibitor (SB203580) (Figures 7B, C). Similarly, IL-8 production induced by EMR2 activation was also reduced by the inhibition of ERK, JNK and IKK but not p38, reconfirming our earlier results (24). Further extensive western blot analyses of 2A1-activated THP-1 cells treated with various signaling inhibitors revealed intricate signaling regulation and potential cross-talk among selective signaling molecules. As such, inhibition of PLC-b (with U73122), PI3K (with Wortmannin and LY294002), and NF-kB activation (with TPCA-1 and Bay11-7082) did not only inactivate Akt, but also ERK phosphorylation. By contrast, inhibition of ERK (with U0126) had no impact on Akt phosphorylation, while inhibition of reactive oxygen species (ROS) production (with NAC) specifically inactivated JNK but  Figure  5C). These results prompted us to propose a Ga 16 -mediated PLC-b dependent signaling network evoked by 2A1-elicited EMR2 activation that worked collectively to induce K + efflux and hence the NLRP3 inflammasome activation signal ( Figure 9).

DISCUSSION
EMR2/ADGRE2 is a human myeloid-restricted aGPCR strongly associated with diverse inflammatory pathologies such as RA, SIRS, and VU (20,33). However, EMR2-mediated signaling events and their functional significance remained to be fully elucidated. In this study, we have unraveled the signaling mechanisms of EMR2 in inducing NLRP3 inflammasome activation in monocytes. The conclusion that EMR2-mediated signaling triggers the activation (2 nd ) signal for NLRP3 inflammasome is reminiscent of other inflammasome-activating GPCRs such as the calcium sensing receptor (CaSR) and its closelyrelated GPRC6A protein (10). Our findings hence confirm and mark EMR2 as one of a limited number of GPCRs known to activate NLRP3 inflammasome in human myeloid cells. EMR2 is a typical aGPCR that undergoes autoproteolytic processing at the extracellular GPCR proteolysis site (GPS) and is expressed as a bipartite complex containing the extracellular Nterminal fragment (NTF) and the seven-transmembrane (7TM) C-terminal fragment (CTF) (34,35). One of the aGPCR activation mechanisms is the tethered-ligand activation model, in which an aGPCR is activated when the NTF dissociates from CTF following the binding of its specific extracellular ligand(s), possibly with the help of mechanical cues (36,37). This allows the exposure of the most N-terminal region of the CTF, called the Stachel peptide, which then acts as a tethered ligand to interact and activate its own 7TM region. Indeed, the EMR2-pC492Y variant has been shown to function as a mechanosensor in mast cells of VU patients and is prone to shed its NTF in response to vibratory challenge (21). As such, it is of great interest to note the compulsory requirement of 2A1 and FHR1 immobilization for efficient EMR2 activation. These results strongly suggested that the multivalent ligation of the receptor molecule by fixed 2A1 and FHR1 ligands likely induced the shedding/dislocation of EMR2-NTF and the subsequent activation of EMR2-CTF in monocytes. Whether EMR2-NTF is shed and, if so, the extent of shedding upon the binding of EMR2 to immobilized 2A1 and FHR1, remain to be characterized. It is noteworthy to mention that the EMR2-pC492Y variant on mast cells is activated by the 2A1 mAb as well as its specific glycosaminoglycan ligand, DS (21,38). The role of glycosaminoglycans in modulating inflammasome activation and inflammatory responses in general has been highlighted by numerous studies (39). For example, the abnormal accumulation of cellular glycosaminoglycans in lysosomal storage disorders was strongly implicated in the pathogenic activation of NLRP3 inflammasome (40). The small proteoglycan, biglycan, was shown to function as a danger signal and induce NLRP3 inflammasome activation by interacting with the TLR2/4 and purinergic P2X4/P2X7 receptors (41). On the other hand, chondroitin sulfate was recently found to attenuate MSU-induced macrophage inflammatory activation in vitro (42). Considering the ubiquitous presence of DS in extracellular matrix and cell surface proteoglycans, the role of EMR2 in modulating NLRP3 inflammasome activation by monocytes in response to quantitative and qualitative changes of DS in tissue microenvironments will be of great interest for a later study.
The identification of a Ga 16 -dependent PLC-b activation pathway induced by 2A1-activated EMR2 is consistent with our earlier findings (24). Importantly, EMR2 coupling to Ga 16 and PLC-b activation was also demonstrated recently by Bhudia et al. who showed that HEK-293T cells co-expressing Ga 16 and EMR2-CTF resulted in inositol monophosphate (IP 1 ) accumulation as well as a strong increase of the NFATluciferase activity, two critical indicators of the activated Ga 16 / PLC-b/Ca 2+ signaling axis (43). While these results reiterate the specific and strong coupling of EMR2 with the Ga 16 protein, we failed to find the involvement of Gbg in 2A1-induced EMR2 signaling, unlike that of FHR1-EMR2 interaction. Interestingly, a recent study by Naranjo et al. similarly revealed that vibratory interaction of DS and EMR2-pC492Y in mast cells induced a Gbg-, Ga q/11 -, and Ga i/o -independent mechanism leading to specific activation of PLC-b, PI3K, ERK1/2 and a transient cytosolic calcium increase (38). One possibility for these differential signaling activities might be due to the potential biased signaling mediated by EMR2 in different cell types (monocyte vs. mast cell) in response to different stimuli (2A1 vs. FHR1 plus NHS vs. DS and vibration). This possibility is further supported by the fact that we did not find a role for NHS in 2A1-induced EMR2 activation ( Figure 5), while it is absolutely required for EMR2 activation elicited by FHR1. The exact role of NHS in FHR1-induced NLRP3 inflammasome activation warrants further investigation. Although most of the downstream signaling effectors of Ga 16 /PLC-b activation, including ERK1/2, JNK, NF-kB, and Akt have been reported in our previous study and re-confirmed here and elsewhere, we further showed that Ca 2+ mobilization, ROS production, and K + efflux are critically involved in the signaling pathways of EMR2-induced NLRP3 inflammasome activation ( Figure 9). While both Ca 2+ and ROS are wellknown triggers of NLRP3 inflammasome, cellular K + efflux has been considered to be the universal inducer of NLRP3 inflammasome activation, involved in the signaling of almost all NLRP3 stimuli, such as ATP, nigericin, and particulate matter (6,8). Hence, we conclude that 2A1-induced EMR2 activation is initiated by Ga 16 coupling and PLC-b stimulation, followed by the activation of Akt, ERK1/2, JNK, NFkB, Ca 2+ mobilization, and ROS production, likely via the generation/ activation of DIG, IP 3 , and PI3K. The collective actions of these signaling events eventually trigger K + efflux and NLRP3 inflammasome activation (Figure 9). It is of great interest to note that PLC-b, Akt, ERK1/2, NFkB, and Ca 2 + mobilization are consistently identified in several independent studies of EMR2 activation and signaling. These signaling intermediates along with EMR2 itself hence represent potential targets of therapeutic intervention for relevant inflammatory disorders.

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. FIGURE 9 | Proposed signaling pathways elicited by 2A1-activated EMR2 receptor that lead to NLRP3 inflammasome activation in monocytic cells. The solid lines indicate positive regulation and the dotted lines represent possible negative regulation/cross talk. ASC, Apoptosis-associated speck-like protein containing a CARD; DAG, diacylglycerol; EMR2, EGF-like module-containing mucin-like hormone receptor-like 2; GAIN, GPCR autoproteolysis-inducing; GPS, GPCR proteolysis site; IP 3 , inositol triphosphate; MAPK, mitogen-activated protein kinase; NLRP3, NOD-, LRR-, and pyrin domain-containing protein 3; PAMP, pathogen-associated molecular pattern; PI3K, phosphoinositide 3-kinase; PLC, phospholipase C; TLRs, toll-like receptors; ROS, reactive oxygen species.