Atherosclerosis, a chronic inflammatory disorder of the walls of arteries, causes more deaths worldwide than any other disease. Cytokines, which are present at high levels in atherosclerotic plaques, play important roles in regulating the initiation and the progression of the disease. Previous studies using animal and cell culture model systems revealed protective, anti-atherogenic effects of the cytokine interleukin-33 (IL-33). The action of this cytokine involves both the induction and suppression of expression of many genes. Unfortunately, the signaling pathways that are responsible for the inhibition of gene expression by this cytokine are poorly understood. Further studies are required given the important roles of genes whose expression is inhibited by IL-33 in key cellular processes associated with atherosclerosis such as monocyte recruitment, foam cell formation and lipoprotein metabolism. We have investigated here the roles of various known IL-33 activated signaling pathways in such inhibitory actions using RNA interference-mediated knockdown assays and monocyte chemotactic protein-1 and intercellular adhesion molecule-1 as model genes. Key roles were identified for extracellular signal-regulated kinase-1/2, p38α kinase, c-Jun N-terminal kinase-1/2, phosphoinositide 3-kinase-γ, and p50 and p65 nuclear factor-κB in such inhibitory action of IL-33. These studies provide new insights on the signaling pathways through which IL-33 inhibits the macrophage expression of key atherosclerosis-associated genes.
Atherosclerosis, a major underlying cause of coronary heart disease (CHD), is associated with inflammation of the arterial wall1,2. The disease is initiated by endothelial cell dysfunction or activation in response to pro-inflammatory stimuli, such as modified low-density lipoproteins (LDL). This results in increased secretion of chemokines [e.g. monocyte chemotactic protein-1 (MCP-1)] by the endothelial cells along with the expression of cell surface adhesion molecules [e.g. intercellular adhesion molecule-1 (ICAM-1)], which together facilitate the recruitment, attachment and infiltration of circulating leukocytes into the subendothelial space1,2. Early atherosclerotic lesions are characterized by the accumulation of macrophages1,2,3. Lesion-resident macrophages take up LDL and oxidized LDL (oxLDL) through several processes, including macropinocytosis, phagocytosis and scavenger receptors-mediated endocytosis, thereby leading to their transformation into lipid-laden foam cells1,2,4,5. Furthermore, macrophages also serve as major sources of pro- and anti-inflammatory cytokines, which play crucial roles in the innate and adaptive immune responses associated with atherosclerosis3,4,6,7. Cytokines participate in all stages of the disease, including endothelial cell dysfunction, foam cell formation and secretion of matrix metalloproteinases involved in the degradation of extracellular matrix components7,8.
Many recent studies have demonstrated a protective anti-atherogenic role for the more recently identified cytokine interleukin (IL)-332,8. Thus, reduced atherosclerosis was seen in the Apolipoprotein E deficient (ApoE−/−) mouse model system following administration of recombinant IL-339. Several mechanisms were identified for the anti-atherogenic effects of IL-33: phenotypic switch from T helper (Th)1 to Th2 cells, increased production of anti-inflammatory cytokines IL-4 and IL-13, and the secretion of oxLDL antibodies9. Conversely, inhibition of IL-33 actions by the administration of a soluble decoy receptor promoted atherosclerosis development in this model system9. In addition, research in our laboratory demonstrated that IL-33 decreased foam cell formation in vivo in such ApoE−/− mice10. Further studies on macrophages, which constitutively express the ST2 receptor10,11,12,13, in vitro revealed the potential mechanisms for the anti-foam cell action of IL-33. Thus, IL-33 caused a reduction in the uptake of modified LDL together with the expression of scavenger receptors SR-A, SR-B1 and CD36 (involved in the cellular uptake of modified LDL) and acyl-coA cholesterol acyltransferase-1 (involved in intracellular esterification of cholesterol)10. In contrast, cholesterol efflux from foam cells was stimulated by IL-33, and associated with up-regulated expression of cholesterol efflux transporters; ATP-binding cassette transporter (ABC)A1 and ABCG110. The requirement of the ST2 receptor in the anti-foam cell actions of IL-33 together with associated changes in gene expression was confirmed by the use of bone marrow-derived macrophages (BMDM) from mice deficient in this receptor10. Subsequent studies have also confirmed the requirement of the ST2 receptor in the IL-33-mediated regulation of expression of other genes in human macrophages together with its anti-foam cell action11,13. IL-33 also inhibited macropinocytosis5, which contributes to the disease via the macrophage uptake of LDL particles1.
Both the up- and down-regulation of gene expression is associated with the cellular actions of IL-33. Several signaling pathways are activated by IL-33 in various cell types, including nuclear factor-κB (NF-κB), phosphoinositide 3-kinase (PI3K) and mitogen activated protein kinases (MAPK)14,15,16,17,18,19,20,21. Unfortunately, in contrast to activation, the signaling pathways involved in the suppression of gene expression by IL-33 are poorly understood mainly due to limited previous research and hence formed the focus of our current study on human macrophages using MCP-1 and ICAM-1 as model genes. We show key roles for extracellular signal-regulated kinase (ERK)-1/2, p38α MAPK, c-Jun N-terminal kinase (JNK)-1/2, PI3K-γ, and p50 and p65 NF-κB.
The expression of MCP-1 and ICAM-1 genes in human macrophages is inhibited by IL-33
We have shown previously that IL-33 attenuates macrophage foam cell formation both in vitro and in vivo10. The action of IL-33 was conserved between human THP-1 macrophages, which have been used in numerous studies on macrophages in relation to atherosclerosis22, and primary cultures of human monocyte-derived macrophages (HMDM) and mouse BMDM, and also extended to the in vivo context10. These studies therefore validate the use of THP-1 macrophages and/or HMDM and/or BMDM for the investigation of IL-33 actions in relation to atherosclerosis10 as employed previously with other agents e.g.23,24,25.
We have previously shown that the expression of scavenger receptors SR-A1, SR-B1 and CD36 was attenuated by IL-33 in both THP-1 macrophages and HMDM10. Preliminary results showed that such an inhibitory action of IL-33 extended to other key atherosclerosis-associated genes: lipoprotein lipase, a key enzyme involved in the control of lipoprotein metabolism26; the adhesion protein ICAM-1, and the chemokines MCP-1, interferon gamma-induced protein-10 and macrophage inflammatory protein-1β in THP-1 macrophages (data not shown). Because of the crucial roles of MCP-1 and ICAM-1 in facilitating the recruitment and attachment of circulating leukocytes during the disease state1,7,8, subsequent studies focused on these atherosclerotic markers with a view of identifying the signaling pathways involved in the suppression of gene expression by the cytokine. Experiments were first carried out on primary cultures of HMDM to confirm that the inhibitory action of IL-33 on MCP-1 and ICAM-1 expression was not because of the use of the THP-1 cell line. As shown in Fig. 1, IL-33 significantly inhibited MCP-1 and ICAM-1 mRNA expression in HMDM (p = 0.040 and p = 0.011, respectively). The concentration of IL-33 used in these experiments (25 ng/ml) was within the physiological range (can reach up to 40 ng/ml)10.
Macrophages, including those derived from THP-1 monocytes, express the ST2 receptor11,12,15,21. For example, our previous studies showed that the ST2 receptor mRNA was expressed in both THP-1 macrophages and HMDM10 and this was extended in the current study to the protein level (Supplementary Fig. 1). Our previous research using BMDM from ST2 deficient mice showed that this receptor was required for all the IL-33-mediated changes in cellular processes and gene expression that we analyzed (e.g. inhibition of modified LDL uptake, cellular levels of total cholesterol and cholesteryl esters and the expression of SR-A1, CD36, SR-B1, ADAMTS-1 and ADAMTS-4; induction of cholesterol efflux and expression of ABCA1, ABCG1 and ApoE)10,13. Nevertheless, a neutralizing antibody with appropriate isotype control was used to investigate the requirement of the ST2 receptor for the changes in the expression of the MCP-1 and ICAM-1 genes by IL-33. Because of technical issues (i.e. compromised IL-33 response in the presence of isotype control antibody for MCP-1), conclusions could only be made for ICAM-1. The inhibition of ICAM-1 expression by IL-33 seen in THP-1 macrophages pre-incubated with the isotype control antibody was attenuated when the cells were instead pre-treated with anti-ST2 neutralizing antibody (Supplementary Fig. 2A). Despite the conservation of responses between THP-1 macrophages and HMDM, experiments were repeated in the latter cellular system. Although the decrease in ICAM-1 expression by IL-33 was not significant in HMDM pre-treated with the isotype control antibody, such a reduction was not seen following pre-treatment of the cells with ST2 neutralizing antibody (Supplementary Fig. 2B). This adds to our previously published studies that shows that the ST2 receptor is required for all the IL-33-mediated changes in cellular processes that we have investigated10,13.
MAP kinases, NF-κB and PI3K-γ are involved in the IL-33-mediated down-regulation of MCP-1 and ICAM-1 expression
IL-33 activates multiple signaling pathways in several cellular systems14,15,16,17,18,19, including PI3K, NF-κB and the three MAPK cascades (ERK, JNK and p38), though very few studies have addressed their roles in relation to inhibition of gene expression by the cytokine. The requirement of key components in the MAPK, NF-κB and PI3Kγ signaling pathways in the inhibition of MCP-1 and ICAM-1 expression by IL-33 was therefore investigated by knockdown assays in THP-1 macrophages. Knockdown of targets was achieved using adenoviral encoding small hairpin RNAs (shRNAs) and/or small interfering RNAs (siRNAs).
The use of shRNA targeting the major p38 isoform, p38α, produced a significant reduction in p38α mRNA and protein levels in vehicle and IL-33 treated cells respectively [55% (p < 0.001) and 50% (p < 0.001) respectively at mRNA expression and 86% (p < 0.001) and 72% (p = 0.002) respectively at the protein level] (Fig. 2A,B). RT-qPCR showed that the significant decrease in MCP-1 and ICAM-1 expression by IL-33 seen in cells transfected with the scramble control (p = 0.008 and p = 0.006, respectively) was attenuated following knockdown of p38α (Fig. 2C,D), thereby indicating a requirement for this kinase in the inhibitory action of this cytokine.
The involvement of the MAPKs JNK1 and JNK2 was investigated by transfection of the cells with siRNAs against JNK-1 and -2. The expression of both isoforms was knocked down together because of the existence of extensive functional redundancy in numerous responses27. The expression of JNK1 mRNA was significantly decreased by 50% in vehicle-treated cells (p = 0.007) with a non-significant reduction (31%; p = 0.130) observed in those stimulated with IL-33 (Fig. 3A). Similarly, JNK2 mRNA expression was significantly decreased by 65% and 31% in vehicle- and IL-33-treated cells respectively (p < 0.001 and p = 0.001 respectively) (Fig. 3B). Western blot analysis confirmed the knockdown at the protein level (Fig. 3C). The significant reduction in MCP-1 and ICAM-1 expression by IL-33 observed in cells transfected with the negative control (p = 0.006 and p = 0.009 respectively) was attenuated following knockdown of JNK1/2 (Fig. 3D,E), with a significant increase in expression seen for MCP-1 (p = 0.016), thereby indicating a requirement for this kinase in the action of IL-33.
For the ERK pathway, the expression was initially knocked down using adenoviral-encoding shRNA against individual isoforms. Because of only a slight reduction of ERK2 at the protein level in IL-33-treated cells (~22%; data not shown), siRNA-mediated knockdown was carried out for this isoform. The data for ERK1 shRNA and ERK2 siRNA are presented in Fig. 4. The knockdown of ERK1 mRNA was 78% and 86% in vehicle- and IL-33-treated cells respectively (p < 0.001 in both cases) and for ERK2 of 40% and 26% respectively (p = 0.001 and 0.168 respectively) (Fig. 4A,B). The knockdown was specific to the isoform (Supplementary Fig. 3A,B). Thus, the expression of ERK1 was not reduced following knockdown of ERK2 and vice versa the expression of ERK2 was not decreased following knockdown of ERK1. Western blot analysis revealed that the knockdown of ERK1 protein was 56% and 48% in vehicle- and IL-33-treated cells respectively (p = 0.009 and p = 0.018 respectively) and for ERK2 of 68% and 64% (p = 0.001 and p = 0.031 respectively) (Fig. 4C,D). Thus, a significant reduction of both ERK1 and -2 proteins was obtained in vehicle- and IL-33-treated cells. Analysis of data from multiple experiments revealed that the knockdown was also specific at the protein level (Supplementary Fig. 3C,D). The significant IL-33-mediated decrease in MCP-1 and ICAM-1 mRNA expression (p = 0.031 and p = 0.005 respectively for ERK1 shRNA, and p = 0.021 and p = 0.004 respectively for ERK2 siRNA) was attenuated following knockdown of ERK-1 or -2 (Fig. 4E–H). In the case of ERK1 knockdown, the reduction in MCP-1 expression by IL-33 was reversed with significantly increased levels being observed (Fig. 4E; p = 0.018). Similarly, in the case of ERK2 knockdown, the IL-33-mediated reduction in ICAM-1 expression was reversed with significantly increased levels also being observed (Fig. 4H; p = 0.039).
p50 and p65 are the major NF-κB family members implicated in cytokine signaling and the control of the inflammatory response2. Their expression was therefore knocked down using siRNA. In the case of p50, a significant decrease in mRNA expression of 55% and 32% was observed in vehicle- and IL-33-treated cells respectively (p < 0.001 and p = 0.022 respectively) (Fig. 5A). For p65, the decrease was 51% and 45% respectively (p < 0.001 in both cases) (Fig. 5B). Western blot analysis confirmed the knockdown at the protein level (Fig. 5C,D). Thus, the knockdown of p50 was 38% and 45% in vehicle- and IL-33-treated cells respectively (p = 0.003 and p < 0.001 respectively) and 56% and 38% respectively in the case of the p65 isoform (p < 0.001 and p = 0.001 respectively) (Fig. 5C,D). In the case of MCP-1, the significant IL-33-mediated reduction of mRNA expression observed in cells transfected with negative control siRNA (p < 0.001) was attenuated following knockdown of both NF-κB isoforms (Fig. 5D). Conclusions could not be made for ICAM-1 expression as none of the changes were significant, including in cells transfected with negative siRNA (data not shown).
The PI3K family member, PI3K-γ, plays an important role in atherosclerosis2. Its involvement was therefore investigated by knockdown using adenoviral-encoding shRNA. The knockdown of PI3K-γ mRNA was 60% and 61% respectively in vehicle- and IL-33 treated cells respectively (p < 0.001 in both cases) (Fig. 6A). The significant reduction in MCP-1 and ICAM-1 expression by IL-33 in cells infected with adenovirus encoding scramble shRNA (p = 0.019 and p = 0.002 respectively) was attenuated following knockdown of PI3K-γ (Fig. 6B,C).
IL-33 is a more recently identified IL-1 family member with important functions in regulating infection, inflammation and cancer21. The actions of this cytokine in such disorders are mediated via a range of immune cells, including macrophages21. We have previously shown inhibition of macrophage foam cell formation in vitro and in vivo by IL-3310. In addition, we demonstrated the requirement of the ST2 receptor for its effect on cholesterol homeostasis and the regulation of gene expression in macrophages in vitro10. We show here that IL-33 attenuates the expression of MCP-1 and ICAM-1, two major pro-atherogenic genes, in human macrophages and identify the roles of key signaling pathways activated by this cytokine in such regulation using knockdown assays.
An important role for IL-33 in atherosclerosis was identified by studies in the ApoE deficient mouse model system9. The disease was attenuated by injection of IL-33 and exacerbated with the soluble ST2 receptor that prevents the cytokine from initiating cellular responses9. IL-33 increased the levels of several anti-atherogenic cytokines such as IL-13, decreased the expression of some pro-atherogenic cytokines (e.g. interferon-γ), caused a Th1 to Th2 shift and increased the concentration of anti-oxLDL antibodies9. However, a recent study in ApoE−/− mice deficient in either IL-33 or its ST2 receptor failed to find any effect on atherosclerosis28. The precise reasons for the discrepancy are currently unclear but differences in the experimental design such as cholesterol levels in the high fat diet and duration of the feeding may have contributed28. In addition, not all the actions of IL-33 are anti-atherogenic; for example, Demyanets et al.29 first showed that IL-33 activates human endothelial cells and increases the expression of MCP-1 and adhesion molecules in these cells in vitro and in human atherosclerotic plaques ex vivo. Subsequently, Pollheimer et al.30 also demonstrated that the cytokine causes endothelial cell activation with stronger responses in nonquiescent cells.
Our studies reveal an anti-atherogenic role for IL-33, provide mechanistic insights into such an action and add to the beneficial effects reported in the prevention of obesity31. For example, IL-33 decreased the formation of macrophage foam cells in vitro and in vivo10. The cytokine attenuated the expression of several key genes involved in modified LDL uptake and intracellular cholesterol storage and simultaneously induced the expression of genes required for the intracellular transport of this sterol and its efflux out of foam cells10. We have also shown that the expression of a disintegrin and metalloproteinase with thrombospondin motifs-4 in human macrophages was inhibited by IL-3313, and recently it has been demonstrated that its deficiency in ApoE−/− mice attenuates atherosclerosis development and improves plaque stability32. The studies presented here show that IL-33 inhibits the expression of MCP-1 and ICAM-1 that have been demonstrated to play pro-atherogenic roles in mouse model systems2,8. Interestingly, IL-33 induces the expression of some of these genes in other cell types; for example, ICAM-1 in human eosinophils33 and MCP-1 and ICAM-1 in human endothelial cells29,30. This implicates cell-type-specific actions of the cytokine that needs to be investigated further. However, the mechanisms are likely to be complex given that the promoter regions of MCP-1 and ICAM-1 genes contain binding sites for several transcription factors (e.g. signal transducer and activator of transcription-1, -3, and -5; E26 transformation-specific; CCAAT/enhancer binding proteins; activator protein-1; specificity protein-1; farnesoid X receptor; peroxisome proliferator-activated receptors etc), many of which have been shown to be functionally important34,35,36,37,38,39,40,41,42,43. All these transcription factors also belong to large families, with both activators and repressors, and additional complexity created by large numbers of post-translational modifications, interactions between them or even with other proteins, and epigenetic regulation36,38,39,40. For example, NF-κB, which is known to stimulate MCP-1 and ICAM-1 expression29,36,40, consists of five members that can also inhibit gene transcription with additional complexity created by interactions with numerous other transcription factors/proteins, post-translational modifications and epigenetic regulation that also produces suppression of gene expression44,45,46,47,48,49,50,51,52. It is therefore not surprising that experiments in mouse models have not always demonstrated pro-atherogenic roles for the different members2.
IL-33 is known to activate several signal transduction pathways such as NF-κB, MAPK and PI3K21. Many studies have determined the signaling pathways underlying IL-33 actions with some cell- and gene-specific responses being identified16,17,18,19,20,53. For example, IL-33 activates p38 MAPK in lung endothelial cells but not in epithelial cells20. The majority of previous research has studied the IL-33-mediated stimulation of gene expression or cellular responses. For instance, IL-33 has been shown to promote ovarian cancer cell growth and metastasis via ERK and JNK signaling pathways54. Unfortunately, the signaling pathways underlying the inhibitory actions of IL-33 are not well understood. Such inhibitory actions extend to key processes such as macrophage foam cell formation and immune cell recruitment to atherosclerotic plaques9,10, decidual natural killer cell cytotoxicity in early human pregnancy55 and cardiac remodelling following myocardial infarction56. Using knockdown assays, we show here a requirement of NF-κB, PI3Kγ and MAPK cascades in the IL-33-mediated inhibition of two key pro-atherogenic genes, MCP-1 and ICAM-1 (Figs 2–6). Interestingly, these pathways are required for both the induction and suppression of gene expression by IL-33. Future studies should investigate how differential responses following activation of these signaling pathways are achieved. These are beyond the scope of current studies because of the immense complexity of IL-33 signaling. For example, data mining has revealed an integrated pathway map of IL-33 and its receptor that consists of 681 proteins and 765 reactions57. The complexity can be gauged by the involvement of 9 transcriptional regulators, 2492 gene regulation events and 740 enzyme catalysis events57. In addition, quantitative phosphoproteomic analysis has revealed IL-33-mediated changes in phosphorylation at 1050 sites in 672 proteins58. Activation of numerous such proteins together with protein-protein interactions may make a key contribution to such differential effects.
In conclusion, the studies presented here supports that IL-33 exerts anti-atherogenic actions. The data demonstrates that the cytokine causes a novel decrease in the expression of MCP-1 and ICAM-1. In addition, key roles for ERK1/2, p38α, JNK1/2, NF-κB and PI3Kγ were identified in such an inhibitory action of the cytokine.
Materials and Methods
Human THP-1 cell line was from Sigma-Aldrich and recombinant human IL-33 was supplied by Peprotech. Antibodies were from Cell Signaling Technology [anti-p44/p42 (ERK1/2) (9102), anti-p38 (9212), anti-JNK1/2 (9252), NF-κB p105/p50 (3035)], Santa Cruz Biotechnology [NF-κB p65 (sc-372), β-Actin (sc-130656)] or Sigma-Aldrich [β-Actin (A2228)].
HMDMs were isolated from buffy coats supplied by the National Blood Service Wales, which were processed immediately following collection using the Ficoll-Hypaque purification method as previously described10,23,25. Informed consent for each donor was granted to the Welsh Blood Service for the use of human blood for non-transfusion purposes10,23,25. All methods were carried out in accordance with the relevant guidelines and regulation (all experimental protocols were approved by the School of Biosciences and Cardiff University)10,23,25. THP-1 and HMDM were cultured in RPMI1640 medium with stable glutamine containing 10% (v/v) heat-inactivated foetal calf serum, penicillin (100 U/ml) and streptomycin (100 µg/ml) at 37 °C in a humidified atmosphere containing 5% (v/v) CO26,13,23,24,25. THP-1 monocytes were differentiated into macrophages by incubation for 24 h with 0.16 µM of phorbol 12-myristate 13-acetate (PMA)6,13,23,24,25.
Real-time quantitative PCR (RT-qPCR)
Isolation of RNA, reverse transcription and RT-qPCR of the resulting cDNA were carried out as described elsewhere6,10,25. The sequences of the primers used are given in Supplementary Table 1. The comparative ΔΔCt method was used to represent relative expression normalized to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH)10,25.
Knockdown assays were performed by transfections using validated siRNAs targeting human ERK2 (SI00300755), JNK1 (SI02758637), JNK2 (SI02222920), p50 (SI02654932) and p65 (SI00301672) from Qiagen. Negative control siRNA (AM4611) was purchased from Invitrogen. Stock solutions of lyophilized siRNA were prepared in nuclease-free water in accordance with the manufacturer’s instructions (Qiagen and Invitrogen).
siRNA transfections were performed in THP-1 monocytes before the addition of PMA for differentiation into macrophages in accordance to the manufacturer’s instructions (Polyplus Transfection) as described in previous studies6,13,25. The preparation of small hairpin RNA (shRNA)-encoding recombinant adenovirus that target ERK-1, ERK-2, p38α or PI3Kγ has been previously described13,24. The adenovirus was then added at a multiplicity of infection of 100 based on previous optimisation studies24,25. The cells were left for 2.5 hours at 37 °C in a humidified incubator containing 5% (v/v) CO2 before the addition of 0.16 µM PMA. Incubation of the cells at 37 °C in a humidified incubator containing 5% (v/v) CO2 was carried out for 48 hours before stimulation with IL-33 or vehicle for the requisite time. Following cytokine stimulation, RNA or protein was isolated as required.
The data are presented as mean ± SEM with normality determined using the Shapiro-Wilk test. Single comparisons were performed using an unpaired Student’s t test, or if the data were not normally distributed using a Mann Whitney U test. The results were regarded as significant where *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001.
The datasets produced and/or analyzed during this study are available from the corresponding author on reasonable request.
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Melanie Buckley was awarded a PhD studentship from the Medical Research Council (Grant G1000385).
The authors declare no competing interests.
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Buckley, M.L., Williams, J.O., Chan, Y. et al. The interleukin-33-mediated inhibition of expression of two key genes implicated in atherosclerosis in human macrophages requires MAP kinase, phosphoinositide 3-kinase and nuclear factor-κB signaling pathways. Sci Rep 9, 11317 (2019) doi:10.1038/s41598-019-47620-8
Molecular Nutrition & Food Research (2019)