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NLRC4 is expressed and inducible in EoL-1 cells. NLRC4 and NAIP mRNA expression in EoL-1 eosinophils treated with TLR2 ligand PAMCSK4 (1 ug/ml) as the first signal and transfected with FLA (100 ng/ml) as the second signal for NLRC4 activation (A). Values represent the mean±SD and experiments were carried out in triplicates. Student's t-test shows the significant difference between stimulated and nonstimulated cells.Immunoblot analysis of EoL-1 cells treated with PAMCSK4 (1 ug/ml) for 4 h and transfected with 100 ng/ml FLA and incubated for another 20 hours (B) or 5 mM ATP for 30 min (C); media was replaced with fresh media and cells were further incubated for another 6 h. EoL-1 cytokine secretion into culture media following PAM3CSK4, PAMCSK4+FLA or PAM3CSK4+ATP treatment was measured by ELISA (D). Cell lysates were prepared and immunoprecipitated with anti-ASC Ab, then followed by precipitation with protein-A/G-magnetic beads. Blots were probed with an anti-NLRC4 Ab and visualized using ECL. Middle panels: controls. Right lane: cell lysates were separated by SDS-PAGE and immunoblotted (E). Human EoL-1 eosinophils were transfected with scrambled siRNA or 3 different siRNAs targeting NLRC4 (S1, S2, S3) or co-transfected with the combination of S1, S2 and S3 siRNAs. A 24 h after transfection, cells were stimulated with NLRC4 agonist and IL-1β secretion was measured by ELISA (F) (scr: scramble siRNA, siRNA cocktail= S1+S2+S3). Immunoblot analysis of cleaved caspase-1 and IL-1β in EoL-1 cells treated with PAMCSK4 and extracellular or transfected FLA (G). Values represent the mean±SD of 2 separate experiments.
Source publication
Eosinophils play critical roles in the maintenance of homeostasis in innate and adaptive immunity. Although primarily known for their roles in parasitic infections and the development of Th2 cell responses, eosinophils also play complex roles in other immune responses ranging from anti-inflammation to defense against viral and bacterial infections....
Contexts in source publication
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... primary eosinophils have already been established (26). Even though NLRC4 is expressed in multiple innate immune cells, its functional role in eosinophils is largely unknown. Quantitative analysis of gene expression and immunoblotting experiments revealed the basal expression of the inflammasome forming molecules NLRC4 and NLRP3 in EoL-1 cells ( Fig. 1). Unlike mice, the human NLRC4 gene has 4 isoforms; therefore, we utilized 2 primer sets to enable detection of all NLRC4 isomers (Fig. 1A). Additionally, we showed that NAIP, ASC and caspase-1, the other members of the NLRC4 inflammasome, are expressed in EoL-1 cells ( Fig. 1B and C). Based on the two-signal model (27)(28)(29), we ...
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... in eosinophils is largely unknown. Quantitative analysis of gene expression and immunoblotting experiments revealed the basal expression of the inflammasome forming molecules NLRC4 and NLRP3 in EoL-1 cells ( Fig. 1). Unlike mice, the human NLRC4 gene has 4 isoforms; therefore, we utilized 2 primer sets to enable detection of all NLRC4 isomers (Fig. 1A). Additionally, we showed that NAIP, ASC and caspase-1, the other members of the NLRC4 inflammasome, are expressed in EoL-1 cells ( Fig. 1B and C). Based on the two-signal model (27)(28)(29), we utilized PAM3CSK4 as the priming signal (signal 1) and FLA from S. typhimirium as the activation signal (signal 2) for NLRC4; PAM3CSK4 (signal ...
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... of the inflammasome forming molecules NLRC4 and NLRP3 in EoL-1 cells ( Fig. 1). Unlike mice, the human NLRC4 gene has 4 isoforms; therefore, we utilized 2 primer sets to enable detection of all NLRC4 isomers (Fig. 1A). Additionally, we showed that NAIP, ASC and caspase-1, the other members of the NLRC4 inflammasome, are expressed in EoL-1 cells ( Fig. 1B and C). Based on the two-signal model (27)(28)(29), we utilized PAM3CSK4 as the priming signal (signal 1) and FLA from S. typhimirium as the activation signal (signal 2) for NLRC4; PAM3CSK4 (signal 1) and ATP (signal 2) were utilized for NLRP3. Stimulation of EoL-1 cells with PAM3CSK4 and FLA led to the upregulation of NLRC4. We then ...
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... (signal 2) for NLRC4; PAM3CSK4 (signal 1) and ATP (signal 2) were utilized for NLRP3. Stimulation of EoL-1 cells with PAM3CSK4 and FLA led to the upregulation of NLRC4. We then performed several experiments to determine if this upregulation of NLRC4 led to formation of functional NLRC4 inflammasomes and recognition of bacterial molecular patterns (Fig. 1B) (30,31). Although, we did not detect any change in NLRP3 and ASC protein expression by immunoblotting, IL-1β and active caspase-1 protein levels were increased after EoL-1 cells were stimulated with PAM3CSK3 and ATP (Fig. 1C). Furthermore, we measured a significant increase in the production of extracellular IL-1β, but not IL-6 ...
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... upregulation of NLRC4 led to formation of functional NLRC4 inflammasomes and recognition of bacterial molecular patterns (Fig. 1B) (30,31). Although, we did not detect any change in NLRP3 and ASC protein expression by immunoblotting, IL-1β and active caspase-1 protein levels were increased after EoL-1 cells were stimulated with PAM3CSK3 and ATP (Fig. 1C). Furthermore, we measured a significant increase in the production of extracellular IL-1β, but not IL-6 cytokines by ELISA after treating EoL-1 cells with either NLRC4 or NLRP3 ligands (signals 1 and 2) ( Fig. 1D), suggesting that NLRC4 and NLRP3 inflammasomes can be activated and are functional in EoL-1 cell line (Fig. ...
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... by immunoblotting, IL-1β and active caspase-1 protein levels were increased after EoL-1 cells were stimulated with PAM3CSK3 and ATP (Fig. 1C). Furthermore, we measured a significant increase in the production of extracellular IL-1β, but not IL-6 cytokines by ELISA after treating EoL-1 cells with either NLRC4 or NLRP3 ligands (signals 1 and 2) ( Fig. 1D), suggesting that NLRC4 and NLRP3 inflammasomes can be activated and are functional in EoL-1 cell line (Fig. ...
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... with PAM3CSK3 and ATP (Fig. 1C). Furthermore, we measured a significant increase in the production of extracellular IL-1β, but not IL-6 cytokines by ELISA after treating EoL-1 cells with either NLRC4 or NLRP3 ligands (signals 1 and 2) ( Fig. 1D), suggesting that NLRC4 and NLRP3 inflammasomes can be activated and are functional in EoL-1 cell line (Fig. ...
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... we confirmed the inflammasome formation by examining the interaction between NLRC4 and ASC adaptor protein by co-IP and detected that endogenous NLRC4 protein precipitated with the ASC after treated with NLRC4 ligands but did not interact with ASC in the non-treated cells (Fig. 1E). Although, IL-1β levels in EoL-1 eosinophils are low, it is biologically relevant and data are consistent with the current literature (7,8). In contrast, IL-33, an important mediator of Th2 responses and eosinophil activation (32), was not secreted by EoL-1 cells at basal levels or upon stimulation (data not shown). To verify that ...
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... stimulation (data not shown). To verify that NLRC4 activation was indeed the reason for the IL-1β secretion upon FLA transfection, NLRC4 mRNA expression was downregulated by RNA interference (RNAi) technology. Human EoL-1 eosinophils transfected with multiple NLRC4-target siRNAs exhibited 45% decrease in NLRC4 mRNA expression by quantitative PCR (Fig. 1E), and this partial NLRC4 knockdown was sufficient to ablate IL-1β secretion, further confirming the NLRC4's role in IL-1β secretion in EoL-1 cells (Fig. 1F). Since bacterial FLA is a cognate ligand of TLR5 (33), we included extracellular FLA in the experimental groups as well to examine whether the cytokine production is via NLRC4. In ...
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... downregulated by RNA interference (RNAi) technology. Human EoL-1 eosinophils transfected with multiple NLRC4-target siRNAs exhibited 45% decrease in NLRC4 mRNA expression by quantitative PCR (Fig. 1E), and this partial NLRC4 knockdown was sufficient to ablate IL-1β secretion, further confirming the NLRC4's role in IL-1β secretion in EoL-1 cells (Fig. 1F). Since bacterial FLA is a cognate ligand of TLR5 (33), we included extracellular FLA in the experimental groups as well to examine whether the cytokine production is via NLRC4. In this case, EoL-1 cells were stimulated with FLA without transfection as TLR5 protein is a transmembrane protein which recognizes its ligand via its ...
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... as well to examine whether the cytokine production is via NLRC4. In this case, EoL-1 cells were stimulated with FLA without transfection as TLR5 protein is a transmembrane protein which recognizes its ligand via its extracellular domain (33). Interestingly, induction through TLR5 did not affect the caspase-1 and IL-1β cleavage and secretion (Fig. ...
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... In contrast, NLRP3 induction had no significant effect on FcεRs and IL-5Rα expression ( Fig. 3A and B). Here, we also included the exctracellularly recognized FLA to further verify the inflammasome dependent proteins, and concluded that not only IL-1β and caspase-1, but also FcεRs are secreted through NLRC4 inflammasome but not TLR5 signaling (Figs. 1G and 3C). These data also suggest that PRRs (NLRC4 and TLR signaling) may impact the interaction between eosinophils and humoral immunity involving B lymphocytes, e.g., the role of IL-6 in aiding the survival of B-cells (37). Furthermore, NLRC4 activation upregulated the human eosinophilic marker Siglec-8 which has an anti-inflammatory function ...
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... primary eosinophils have already been established (26). Even though NLRC4 is expressed in multiple innate immune cells, its functional role in eosinophils is largely unknown. Quantitative analysis of gene expression and immunoblotting experiments revealed the basal expression of the inflammasome forming molecules NLRC4 and NLRP3 in EoL-1 cells ( Fig. 1). Unlike mice, the human NLRC4 gene has 4 isoforms; therefore, we utilized 2 primer sets to enable detection of all NLRC4 isomers (Fig. 1A). Additionally, we showed that NAIP, ASC and caspase-1, the other members of the NLRC4 inflammasome, are expressed in EoL-1 cells ( Fig. 1B and C). Based on the two-signal model (27)(28)(29), we ...
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... in eosinophils is largely unknown. Quantitative analysis of gene expression and immunoblotting experiments revealed the basal expression of the inflammasome forming molecules NLRC4 and NLRP3 in EoL-1 cells ( Fig. 1). Unlike mice, the human NLRC4 gene has 4 isoforms; therefore, we utilized 2 primer sets to enable detection of all NLRC4 isomers (Fig. 1A). Additionally, we showed that NAIP, ASC and caspase-1, the other members of the NLRC4 inflammasome, are expressed in EoL-1 cells ( Fig. 1B and C). Based on the two-signal model (27)(28)(29), we utilized PAM3CSK4 as the priming signal (signal 1) and FLA from S. typhimirium as the activation signal (signal 2) for NLRC4; PAM3CSK4 (signal ...
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... of the inflammasome forming molecules NLRC4 and NLRP3 in EoL-1 cells ( Fig. 1). Unlike mice, the human NLRC4 gene has 4 isoforms; therefore, we utilized 2 primer sets to enable detection of all NLRC4 isomers (Fig. 1A). Additionally, we showed that NAIP, ASC and caspase-1, the other members of the NLRC4 inflammasome, are expressed in EoL-1 cells ( Fig. 1B and C). Based on the two-signal model (27)(28)(29), we utilized PAM3CSK4 as the priming signal (signal 1) and FLA from S. typhimirium as the activation signal (signal 2) for NLRC4; PAM3CSK4 (signal 1) and ATP (signal 2) were utilized for NLRP3. Stimulation of EoL-1 cells with PAM3CSK4 and FLA led to the upregulation of NLRC4. We then ...
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... (signal 2) for NLRC4; PAM3CSK4 (signal 1) and ATP (signal 2) were utilized for NLRP3. Stimulation of EoL-1 cells with PAM3CSK4 and FLA led to the upregulation of NLRC4. We then performed several experiments to determine if this upregulation of NLRC4 led to formation of functional NLRC4 inflammasomes and recognition of bacterial molecular patterns (Fig. 1B) (30,31). Although, we did not detect any change in NLRP3 and ASC protein expression by immunoblotting, IL-1β and active caspase-1 protein levels were increased after EoL-1 cells were stimulated with PAM3CSK3 and ATP (Fig. 1C). Furthermore, we measured a significant increase in the production of extracellular IL-1β, but not IL-6 ...
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... upregulation of NLRC4 led to formation of functional NLRC4 inflammasomes and recognition of bacterial molecular patterns (Fig. 1B) (30,31). Although, we did not detect any change in NLRP3 and ASC protein expression by immunoblotting, IL-1β and active caspase-1 protein levels were increased after EoL-1 cells were stimulated with PAM3CSK3 and ATP (Fig. 1C). Furthermore, we measured a significant increase in the production of extracellular IL-1β, but not IL-6 cytokines by ELISA after treating EoL-1 cells with either NLRC4 or NLRP3 ligands (signals 1 and 2) ( Fig. 1D), suggesting that NLRC4 and NLRP3 inflammasomes can be activated and are functional in EoL-1 cell line (Fig. ...
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... by immunoblotting, IL-1β and active caspase-1 protein levels were increased after EoL-1 cells were stimulated with PAM3CSK3 and ATP (Fig. 1C). Furthermore, we measured a significant increase in the production of extracellular IL-1β, but not IL-6 cytokines by ELISA after treating EoL-1 cells with either NLRC4 or NLRP3 ligands (signals 1 and 2) ( Fig. 1D), suggesting that NLRC4 and NLRP3 inflammasomes can be activated and are functional in EoL-1 cell line (Fig. ...
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... with PAM3CSK3 and ATP (Fig. 1C). Furthermore, we measured a significant increase in the production of extracellular IL-1β, but not IL-6 cytokines by ELISA after treating EoL-1 cells with either NLRC4 or NLRP3 ligands (signals 1 and 2) ( Fig. 1D), suggesting that NLRC4 and NLRP3 inflammasomes can be activated and are functional in EoL-1 cell line (Fig. ...
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... we confirmed the inflammasome formation by examining the interaction between NLRC4 and ASC adaptor protein by co-IP and detected that endogenous NLRC4 protein precipitated with the ASC after treated with NLRC4 ligands but did not interact with ASC in the non-treated cells (Fig. 1E). Although, IL-1β levels in EoL-1 eosinophils are low, it is biologically relevant and data are consistent with the current literature (7,8). In contrast, IL-33, an important mediator of Th2 responses and eosinophil activation (32), was not secreted by EoL-1 cells at basal levels or upon stimulation (data not shown). To verify that ...
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... stimulation (data not shown). To verify that NLRC4 activation was indeed the reason for the IL-1β secretion upon FLA transfection, NLRC4 mRNA expression was downregulated by RNA interference (RNAi) technology. Human EoL-1 eosinophils transfected with multiple NLRC4-target siRNAs exhibited 45% decrease in NLRC4 mRNA expression by quantitative PCR (Fig. 1E), and this partial NLRC4 knockdown was sufficient to ablate IL-1β secretion, further confirming the NLRC4's role in IL-1β secretion in EoL-1 cells (Fig. 1F). Since bacterial FLA is a cognate ligand of TLR5 (33), we included extracellular FLA in the experimental groups as well to examine whether the cytokine production is via NLRC4. In ...
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... downregulated by RNA interference (RNAi) technology. Human EoL-1 eosinophils transfected with multiple NLRC4-target siRNAs exhibited 45% decrease in NLRC4 mRNA expression by quantitative PCR (Fig. 1E), and this partial NLRC4 knockdown was sufficient to ablate IL-1β secretion, further confirming the NLRC4's role in IL-1β secretion in EoL-1 cells (Fig. 1F). Since bacterial FLA is a cognate ligand of TLR5 (33), we included extracellular FLA in the experimental groups as well to examine whether the cytokine production is via NLRC4. In this case, EoL-1 cells were stimulated with FLA without transfection as TLR5 protein is a transmembrane protein which recognizes its ligand via its ...
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... as well to examine whether the cytokine production is via NLRC4. In this case, EoL-1 cells were stimulated with FLA without transfection as TLR5 protein is a transmembrane protein which recognizes its ligand via its extracellular domain (33). Interestingly, induction through TLR5 did not affect the caspase-1 and IL-1β cleavage and secretion (Fig. ...
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... In contrast, NLRP3 induction had no significant effect on FcεRs and IL-5Rα expression ( Fig. 3A and B). Here, we also included the exctracellularly recognized FLA to further verify the inflammasome dependent proteins, and concluded that not only IL-1β and caspase-1, but also FcεRs are secreted through NLRC4 inflammasome but not TLR5 signaling (Figs. 1G and 3C). These data also suggest that PRRs (NLRC4 and TLR signaling) may impact the interaction between eosinophils and humoral immunity involving B lymphocytes, e.g., the role of IL-6 in aiding the survival of B-cells (37). Furthermore, NLRC4 activation upregulated the human eosinophilic marker Siglec-8 which has an anti-inflammatory function ...
Similar publications
Toll-like receptor 5 (TLR5) is a pattern recognition receptor that specifically recognizes flagellin and consequently plays a crucial role in the control of intestinal homeostasis by activating innate and adaptive immune responses. TLR5 overexpression, on the other hand, might disrupt the intestinal mucosal barrier, which serves as the first line o...
Citations
... Previous study showed that activation of the NLRC4 inflammasome leads to maturation of caspase-1, then mediates the excessive secretion of proinflammatory cytokines such as interleukin-1b (IL-1b), IL-1β and IL-18 [16,17]. Strikingly, activation of the NLRC4 inflammasome also promoted expression of the costimulatory receptor CD80 as well as expression of immunoregulatory receptors PD-L1 and Siglec-8 [18]. This likely represents a major pathway by which reduced NLRC4 methylation promotes systemic inflammation in KD. ...
Background
Kawasaki disease (KD) is a systemic vasculitis accompanied by many systemic physiological and biochemical changes. Elucidating its molecular mechanisms is crucial for diagnosing and developing effective treatments. NLR Family CARD Domain Containing 4 (NLRC4) encodes the key components of inflammasomes that function as pattern recognition receptors. The purpose of this study was to investigate the potential of NLRC4 methylation as a biomarker for KD.
Methods
In this study, pyrosequencing was utilized to analyze NLRC4 promoter methylation in blood samples from 44 children with initial complete KD and 51 matched healthy controls. Methylation at five CpG sites within the NLRC4 promoter region was evaluated.
Results
Compared to controls, NLRC4 methylation significantly decreased in KD patients (CpG1: p = 2.93E-06; CpG2: p = 2.35E-05; CpG3: p = 6.46E-06; CpG4: p = 2.47E-06; CpG5: p = 1.26E-05; average methylation: p = 5.42E-06). These changes were significantly reversed after intravenous immunoglobulin (IVIG) treatment. ROC curve analysis demonstrated remarkable diagnostic capability of mean NLRC4 gene methylation for KD (areas under ROC curve = 0.844, sensitivity = 0.75, p = 9.61E-06, 95% confidence intervals were 0.762–0.926 for mean NLRC4 methylation). In addition, NLRC4 promoter methylation was shown to be significantly negatively correlated with the levels of central granulocyte percentage, age, mean haemoglobin quantity and mean erythrocyte volume. Besides, NLRC4 promoter methylation was positively correlated with lymphocyte percentage, lymphocyte absolute value.
Conclusions
Our work revealed the role of peripheral NLRC4 hypomethylation in KD pathogenesis and IVIG treatment response, could potentially serve as a treatment monitoring biomarker, although its precise functions remain to be elucidated.
... Heretofore, most biological activities of IL-18BP were focused on suppressing Th1 immunity to treat Th1 autoimmune diseases. However, it is necessary to investigate the role of IL-18BP in Th2 autoimmune disease since recent study suggested the potential role of NLR family CARD domain-containing protein 4 (NLRC4), which is known for IL-18 processing enzyme, mediating a variety of eosinophilic Th2 immune response (11). In addition to this, alarmins induce a Th2 immune response that triggers the alarm of IL-33, IL-25, and TSLP was first discovered in allergies and infectious diseases (12). ...
IL-18 binding protein (IL-18BP) was originally discovered in 1999 while attempting to identify an IL-18 receptor ligand binding chain (also known as IL-18Rα) by subjecting concentrated human urine to an IL-18 ligand affinity column. The IL-18 ligand chromatography purified molecule was analyzed by protein microsequencing. The result revealed a novel 40 amino acid polypeptide. To isolate the complete open reading frame (ORF), various human and mouse cDNA libraries were screened using cDNA probe derived from the novel IL-18 affinity column bound molecule. The identified entire ORF gene was thought to be an IL-18Rα gene. However, IL-18BP has been proven to be a unique soluble antagonist that shares homology with a variety of viral proteins that are distinct from the IL-18Rα and IL-18Rβ chains. The IL-18BP cDNA was used to generate recombinant IL-18BP (rIL-18BP), which was indispensable for characterizing the role of IL-18BP in vitro and in vivo. Mammalian cell lines were used to produce rIL-18BP due to its glycosylation-dependent activity of IL-18BP (approximately 20 kDa). Various forms of rIL-18BP, intact, C-terminal his-tag, and Fc fusion proteins were produced for in vitro and in vivo experiments. Data showed potent neutralization of IL-18 activity, which seems promising for clinical application in immune diseases involving IL-18. However, it was a long journey from discovery to clinical use although there have been various clinical trials since IL-18BP was discovered in 1999. This review primarily covers the discovery of IL-18BP along with how basic research influences the clinical development of IL-18BP.
... The NAIP-NLRC4 inflammasomes recognize Gram-negative bacteria such as Salmonella typhimurium, Pseudomonas aeruginosa, and Legionella pneumophila [21,22]. In one of our recent studies, we reported the involvement of NLRC4 in various eosinophilic functions through the induction of surface molecules, including CD63, CD80, and FcεRII [23]. ...
... in TLR2-Primed EoL-1 Cells In Vitro. We used EoL-1 human eosinophilic leukemia cell line as a model for the investigation of eosinophilic functions for four main reasons: (1) their rapid (within hours) responsiveness to broad range of stimuli, (2) their expression profile of PRRs whose roles in regulating eosinophilic responses are largely unknown, (3) their ability to express human eosinophilic pan markers, including Siglec-8 and IL5R [23,24], and (4) eosinophils' sparsity in blood. Our previous study already exhibited that NLRC4 is expressed and inducible in EoL-1 cells at both the mRNA levels and protein levels [23]. ...
... We used EoL-1 human eosinophilic leukemia cell line as a model for the investigation of eosinophilic functions for four main reasons: (1) their rapid (within hours) responsiveness to broad range of stimuli, (2) their expression profile of PRRs whose roles in regulating eosinophilic responses are largely unknown, (3) their ability to express human eosinophilic pan markers, including Siglec-8 and IL5R [23,24], and (4) eosinophils' sparsity in blood. Our previous study already exhibited that NLRC4 is expressed and inducible in EoL-1 cells at both the mRNA levels and protein levels [23]. Since both Ag-specific IgE and eosinophils are the participants of the Th2-mediated allergic inflammation, we used Ova and Ova-specific IgE to form Ova-IgE ICs. ...
Immune complexes (ICs) skew immune responses toward either a pro- or anti-inflammatory direction based on the type of stimulation. Immunoglobulin E (IgE) is associated with Th2 immune responses and known to activate innate immune cells. However, roles of antigen (Ag)-specific-IgE ICs in regulating human eosinophil responses remain elusive; therefore, this study builts upon the mechanism of which ovalbumin (Ova)-IgE ICs affects eosinophilic responses utilizing human EoL-1 cell line as a model. Eosinophils are granulocytes functioning through pattern recognition receptors (PRRs) and destructive granule contents in allergic inflammation and parasitic infections. One of the PRRs that eosinophils express is NLRC4, a member of the CARD domain containing nucleotide-binding oligomerization (NOD)-like receptor (NLR) family. Upon recognition of its specific ligand flagellin, NLRC4 inflammasome is formed and leads to the release of interleukin-1β (IL-1β). We exhibited that Ova-IgE ICs induced the NLRC4-inflammasome components, including NLRC4, caspase-1, intracellular IL-1β, and secretion of IL-1β, as well as the granule contents MMP9, TIMP1, and TIMP2 proteins via TLR2 signaling; these responses were suppressed, when NLRC4 inflammasome got actived in the presence of ICs. Furthermore, Ova-IgE ICs induced mRNA expressions of MMP9, TIMP2, and ECP and protein expressions of MMP9 and TIMP2 in EoL-1 through FcɛRII. Interestingly, TLR2 ligand and Ova-IgE ICs costimulation elevated the number of CD63+ cells, a degranulation marker, as compared to the native IgE. Collectively, our findings provide a mechanism for the impacts of Ova-IgE ICs on eosinophilic responses via NLRC4-inflammasome and may help understand eosinophil-associated diseases, including chronic eosinophilic pneumonia, eosinophilic esophagitis, eosinophilic granulomatosis, parasitic infections, allergy, and asthma.
... Indeed, our knowledge of how PRRs trigger these reactions has greatly increased for the past few decades. Several studies have reported the expression and importance of TLRs, NLRs, RLRs and CLRs in eosinophils (11,(13)(14)(15) suggesting that these receptors may be responsible for the development of EADs including eosinophilic asthma, chronic rhinosinusitis, idiopathic eosinophilia, eosinophilic leukemia, helminth infections and rare conditions such as HES and eosinophilic gastrointestinal disorders (EGIDs) (16). Thus, we analyzed the PRR expression profiles of EoL-1 and HL-60 human eosinophil like cell lines and compared them to primary eosinophils in silico. ...
... We previously showed that EoL-1 cells express NLRP3 and NLRC4 as well as other inflammasome components such as caspase-1, ASC and NAIP at both mRNA and protein levels. Stimulation of EoL-1 cells with TLR2 ligand PAM3CSK4 and TLR5 ligand Flagella activated EoL-1 cells and triggered the formation of NLRC4 inflammasome which led to IL-1β secretion (15). Furthermore, according to the CCLE cell line gene expression profiles database, EoL-1 cells can express NOD1 and NOD2. ...
... Given that immune system components can regulate autophagy through immune cells and cytokine release to protect the cells [37], we assessed the inflammatory response after induction of EoL-1 cells with VPA. We already reported that eosinophils can induce inflammatory responses through the CARD domain-containing NOD-like receptor 4 (NLRC4) which forms multimeric protein complexes called "inflammasomes" [38]. NLR proteins are members of the pattern recognition receptor (PRR) families and are known to function as NLR proteins without the formation of inflammasomes. ...
... Unfortunately, the low numbers of human eosinophils in circulating blood and the insufficient protein and mRNA yield from primary human eosinophils generate technical challenges for mechanistic studies, which require high concentrations of cellular products. Therefore, we adapted the human EoL-1 eosinophil cell line as a model due to their ability to quickly respond to wide range of stimulants and for their human eosinophilic pan-marker expression profiles [20,38]. In addition, human eosinophilic cell lines, including EoL-1, its derivative EoL-3, HL-60, AML-14, and its subclone AML14.3D10, are other available cell lines for human eosinophil studies. ...
... The primers that were used for quantitative real-time RT-PCR were hNrf2 primers (F 5 -GAGAGCCCAGTCTTCATTGC-3 , R 5 -TGCTCAATGTCCTGTTGCAT-3 ), hNLRC4 primers (F 5 -GTGTTCTCCCACAAGTTTGA-3 , R 5 -AGTAACCATTCCCCTTGGTC-3 ), hNLRP3 primers (F 5 -CTTCCTTTCCAGTTTGCTGC-3 , R 5 -TCTCGCAGTCCACTTCCTTT-3 ) and hHPRT 1 primers (F 5 -GACCAGTCAACAGGGGACAT-3 , R 5 -AACACTTCGTGGG GTCCTTTTC-3 ) as housekeeping genes using QuantiTect SYBR Green RT-PCR (Qiagen, Waltham, MA, USA) to determine mRNA expression levels. Each RT-PCR reaction was performed as previously described [38,51,52]. The mRNA levels for the target gene were corrected to those of the housekeeping gene (HPRT) and then were calculated by subtracting their corresponding cycle threshold (Ct) before and after stimulation using the following formula: ...
Eosinophils function in rapid innate immune responses and allergic reactions. Recent research has raised the possibility that the histone deacetylase inhibitor valproic acid (VPA) may be a promising therapeutic agent for treatment of allergic responses and certain cancers. However, its effects on eosinophils remain unclear. Utilizing the EoL-1 human eosinophil cell line as a model, we investigated the effects of VPA on oxidative stress- and autophagy-mediated immune responses. We found that VPA induced reactive oxidative species (ROS) generation and eosinophil activation without affecting cell viability. Moreover, VPA treatment suppressed the negative regulator of antioxidant transcription factor Nrf2, which is known to activate antioxidant defense. Interestingly, VPA was able to increase autophagic markers, as well as NLRP3 and NLRC4 mRNA activation, in Eol-1 cells in a dose-dependent manner. Collectively, our results indicate that VPA could increase the severity of allergic responses, and if so, it clearly would not be a suitable drug for the treatment of allergic reactions. However, VPA does have the potential to induce autophagy and to regulate the inflammatory responses via inflammasome-driven caspase-1 deactivation in a dose-dependent manner.
... NSHL constitutes the most classical Hodgkin lymphoma, with a higher frequency in younger patients. An inflammatory infiltrate rich in eosinophils is not rare in NSHL [32]. Eosinophils possess the biological role of upregulating the expression of PD-L1 via NLR Family CARD Domain Containing 4 (NLRC4) inflammasome-mediated action and promoting the secretion of a proliferation-inducing ligand (APRIL) [33,34] (Supplemental Figure S1). ...
Kimura’s disease (KD) is a rare lymphoproliferative fibroinflammatory disorder that commonly affects the subcutaneous tissue and lymph nodes of the head and neck. The condition is a reactive process involving T helper type 2 cytokines. Concurrent malignancies have not been described. Differential diagnosis with lymphoma can be challenging without tissue biopsy. Here, we present the first reported case of coexisting KD and eosinophilic nodular sclerosis Hodgkin lymphoma of the right cervical lymphatics in a 72-year-old Taiwanese man.
The innate immune response represents the first line of host defense, and it is able to detect pathogen- and damage-associated molecular patterns (PAMPs and DAMPs, respectively) through a variety of pattern recognition receptors (PRRs). Among these PRRs, certain cytosolic receptors of the NLRs family (specifically NLRP1, NLRP3, NLRC4, and NAIP) or those containing at least a pyrin domain (PYD) such as pyrin and AIM2, activate the multimeric complex known as inflammasome, and its effector enzyme caspase-1. The caspase-1 induces the proteolytic maturation of the pro-inflammatory cytokines IL-1ß and IL-18, as well as the pore-forming protein gasdermin D (GSDMD). GSDMD is responsible for the release of the two cytokines and the induction of lytic and inflammatory cell death known as pyroptosis. Each inflammasome receptor detects specific stimuli, either directly or indirectly, thereby enhancing the cell's ability to sense infections or homeostatic disturbances. In this chapter, we present the activation mechanism of the so-called "canonical" inflammasomes.
Inflammasomes, composed of the nucleotide-binding oligomerization domain(NOD)-like receptors (NLRs), are immune-functional protein multimers that are closely linked to the host defense mechanism. When NLRs sense pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), they assemble into inflammasomes. Inflammasomes can activate various inflammatory signaling pathways, including nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways, and produce a large number of proinflammatory cytokines, which are closely associated with multiple cancers. They can also accelerate the occurrence and development of cancer by providing suitable tumor microenvironments, promoting tumor cell proliferation, and inhibiting tumor cell apoptosis. Therefore, the exploitation of novel targeted drugs against various inflammasomes and proinflammatory cytokines is a new idea for the treatment of cancer. In recent years, more than 50 natural extracts and synthetic small molecule targeted drugs have been reported to be in the research stage or have been applied to the clinic. Herein, we will overview the mechanisms of inflammasomes in common cancers and discuss the therapeutic prospects of natural extracts and synthetic targeted agents.
Objectives:
MiR-223-3p is a multifunctional microRNA regulated by multiple transcription factors and plays a critical role in inflammation. This paper was designed to investigate the regulatory role and mechanism of miR-223-3p in eosinophils degranulation and allergic rhinitis (AR) inflammation.
Methods:
OVA sensitized AR mouse model and EOL-1 cells model were established. RT-qPCR and FISH were performed to detect the miR-223-3p expression. ELISA and WB were utilized to evaluate mRNA and protein expression. HE staining and transmission electron microscopy were applied to observe the morphological changes in nasal mucosa. Flow cytometry and immunofluorescence staining were performed to measure the proportion of eosinophils and eosinophilic major basic protein expression. The targeting relationship between miR-223-3p and FBXW7 was verified by bioinformatic analysis and dual-luciferase reporter gene assay. The expression of FBXW7 was detected by immunohistochemistry.
Results:
The level of miR-223-3p in nasal mucosa was significantly up-regulated in AR group. The expression of miR-223-3p, ECP, MBP, and EPO were increased in EOL-1 cells, further increasing the miR-223-3p level could promote the ECP and EPO mRNA expression. Upregulation of miR-223-3p increased eosinophils granule protein expression, aggravated mucosal destruction and enhanced AR inflammation. Luciferase assay verified miR-223-3p directly target the 3'-UTR of FBXW7. In vitro, overexpression of FBXW7 could reverse the increase in MBP expression caused by the up-regulation of miR-223-3p. In vivo, knockdown of FBXW7 could reverse the down-regulation in granule protein level caused by the down-regulation of miR-223-3p, thereby aggravating AR inflammation.
Conclusion:
Collected evidence elucidated that miR-223-3p could regulate the eosinophil degranulation and enhances the inflammation in AR by targeting FBXW7. The miR-223-3p/FBXW7 axis may provide a novel approach for AR treatment.
Background:
Gram-negative sepsis is closely related to the immune response, involving collaborative efforts of different immune cells. However, the mechanisms underlying immune cell regulation in gram-negative sepsis remain unclear. Therefore, this study investigated the potential regulatory mechanisms and identified the key genes related to immune cells in gram-negative sepsis.
Methods:
The RNA-sequencing data for gram-negative sepsis samples and normal samples were collected from the Gene Expression Omnibus (GEO) dataset GSE9960. CIBERSORT was performed to analyze the proportion of 22 types of immune cells in gram-negative sepsis and normal samples. Weighted gene co-expression network analysis (WGCNA) was used to determine the networks that are associated with the differentially distributed immune cells in the two groups. Differentially expressed genes were identified using the limma package. The least absolute shrinkage and selection operator (LASSO) and support vector machine-recursive feature elimination (SVM-RFE) algorithms were applied to ascertain hub gene signatures. The gene interaction network of hub gene signatures was determined by ingenuity pathway analysis. Furthermore, the expression levels of the key genes were verified using quantitative real-time polymerase chain reaction (qRT-PCR).
Results:
CIBERSORT analysis showed that the proportions of plasma cells, resting CD4+ memory T cells, M1 macrophages, and eosinophils were significantly different between gram-negative sepsis and normal samples. WGCNA identified 1,100 genes in the most relevant modules associated with these immune cells. In addition, 87 differentially expressed genes were identified. By overlapping the genes found in the WGCNA and the differentially expressed genes, a total of 46 genes related to immune cells were identified. Integrative analysis of LASSO and SVM-RFE identified NLR family CARD domain-containing 4 (NLRC4) and ral guanine nucleotide dissociation stimulator like 4 (RGL4) as key gene signatures related to immune cells in gram-negative sepsis. The qRT-PCR results demonstrated that both NLRC4 and RGL4 were upregulated in peripheral blood mononuclear cells (PBMCs) from patients with sepsis.
Conclusions:
This investigation provides novel insights into the molecular mechanisms of immune cells involved in the pathogenesis of gram-negative sepsis. NLRC4 and RGL4 were identified as key gene signatures related to immune cells and may act as potential diagnostic biomarkers for gram-negative sepsis.
