ST2 and IL33

ST2 is a member of the IL-1R subfamily and was identified in 1989 [1]. The ST2 gene is located on chromosome 2q12 in humans and contains 13 introns and spans 40 kilobases (kb). In humans three splice variants exist, ST2L, sST2 and ST2V (Figure 1a). ST2L is a transmembrane bound receptor and as such specifically belongs to the Toll/IL-1R (TIR) superfamily and shows homology to the intracellular domain of IL-1R1 [2]. sST2 is a soluble protein with no transmembrane sequence, it is excreted extracellularly and binds to IL-33. Both ST2L and sST2 contain three identical Ig extracellular domains, although sST2 lacks the transmembrane sequence. Instead sST2 contains an additional 9 amino acids at the C terminus. sST2 is thought to act as a decoy receptor sequestering IL-33 away from the transmembrane bound receptor ST2L. ST2V is a membrane-bound receptor that contains a hydrophobic tail. It contains two Ig domains and is expressed in the gut [3]. The function of ST2V has not been fully elucidated.

All ST2 isoforms are produced from the IL-1RL1 gene as a result of alternative splicing under the control of two distinct promoters, the distal and the proximal promoter (Figure 1b) [4]. Early ST2 expression studies were carried out on the rat ST2 gene, known as Fit1.

Figure 1: There are three human ST2 isoforms- ST2L, sST2 and ST2V. ST2 is encoded by the IL1RL1 gene. In humans three splice variants exist, ST2L, sST2 and ST2V. (a) ST2L is a transmembrane receptor that binds to IL-33. sST2 is a soluble protein with no transmembrane sequence and can also bind to IL-33. ST2V is a membrane-bound receptor that contains two Ig domains and a hydrophobic tail. (b) All ST2 isoforms are produced from the IL-1RL1 gene as a result of alternative splicing under the control of two distinct promoters, i.e. the distal and the proximal promotor

The ST2 gene contains two discrete promoters. It was originally suggested that the sST2 and ST2L mRNA isoforms were transcribed from different promoters resulting in alternative splicing to generate two discrete 3’ coding sequences. Further investigations demonstrated that both the human and murine ST2 genes similarly contain both promoters, a distal and a proximal promoter [104]. However, in contrast to the initial study, it was shown that the two isoforms can be transcribed from either promoter, with the choice of promoter governed by the cell type. For instance, the human leukaemic cell line, UT-7 can transcribe both ST2 isoforms using either the distal or proximal promoter, but predominantly use the distal promoter. In contrast, most ST2 expression in the human TM12 fibroblastic cell line is initiated from the proximal promoter. Similarly, it was found that mast cells utilised the distal promoter, while fibroblasts employed the proximal promoter, suggesting that promoter usage is dependent on the cell type and is not transcript specific.

The distal ST2 promoter contains two GATA consensus sites which enable binding of the transcription factors GATA1 and GATA2, both of which are involved in the regulation of ST2 gene expression. These transcription factors bind upstream of the transcription start site and regulate ST2 gene transcription. The ST2 promoter is transactivated by GATA-2 and repressed by GATA-1 in mast cells and basophils [5]. In mast cells and Th2 cells, ST2L expression is regulated by transcription factors GATA-3 and STAT5 , while the transcription factor Gfi1 was found to upregulate surface expression of ST2L in innate lymphoid cells. IL-33, the ligand for ST2, has been found to downregulate sST2 and ST2L mRNA in pancreatic cancer cells [6].

The kidney, lung, placenta and stomach all express high levels of sST2 and ST2L. In addition, many endothelial cells from lung, bronchus, coronary artery and umbilical cord express both ST2L and sST2 mRNA [7]. The expression levels of the isoforms vary, with ST2L expression found to be higher in the spleen, heart, testis and colon than sST2, whilst sST2 expression levels are higher in the brain and liver. sST2 is induced by serum. ST2L is also expressed on the surface of fibroblasts and hematopoietic cells such as T helper type 2 (Th2) lymphocytes and mast cells, and has recently been found to be expressed by Th1 cells, CD8+T cells, NK cells and NKT cells [8].

IL-33 was originally named nuclear factor from high endothelial venules (NF-HEV), as it was first found to be expressed in the nucleus of quiescent endothelial cells [9]. Later, in 2005, IL-33 was identified as a member of the IL-1 family of cytokines and as the ligand for ST2L. Unlike other IL-1 family members, which are located on chromosome 2, the IL-33 gene is located on chromosome 9 at 9p24.1. It spans ~16 kb and contains seven exons and contains two alternative promoters. Human IL-33 comprises 270 amino acids (aa), while murine IL-33 consists of 266 aa .

Different IL-33 splice variants have been observed in human tissues. IL-33 is generated as a full-length protein [10], and contains a caspase-1 cleavage site. It was originally proposed that full-length IL-33 required cleavage by caspase-1 [125], similar to IL-1β and IL-18, to generate the mature biologically active form of the cytokine [11]. However, further studies demonstrated that full-length IL-33 does not require activation by caspase-1 to be active. Although IL-33 is not activated by caspase-1, IL-33 can be processed by caspase-3 and caspase-7 to yield biologically inactive fragments [12].

Processing by proteolytic enzymes may play a vital part in modulating IL-33 activity during inflammation. Un-cleaved full-length IL-33 is released from the nucleus of barrier tissue during necrosis and functions as a danger signal or ‘alarmin’. However, the alarmin signal can be amplified by cleavage of macrophage, neutrophil, or mast cell-derived proteases, such as elastase, which cleaves full-length IL-33 into the IL-3395-270, IL-3399-270, and IL-33109-270 mature forms. These isoforms are between 18 and 21 kDa in human and 20 kDa in mouse, i.e. IL-33102-266. These mature forms of IL-33 have up to 30-fold increased biological activity compared to the full length uncleaved, IL-33 [13]. In this way a small number of alarmin molecules can have a potent local effect upon proteolytic cleavage of the N-terminus. Bioactivity is lost upon cleavage of the core IL-1 family structure by chymase or other proteases secreted by mast cells, thus silencing the alarmin signal. A second mechanism of silencing IL-33 has also been documented. This involves the formation of two disulphide bridges, which inhibits binding of IL-33 to ST2L, thus abrogating the ability of IL-33 to transduce a signal.

IL-33 is expressed by a diverse range of cells in many organs, such as activated leukocytes, especially innate immune cells (e.g. mast cells, macrophages and DCs), endothelial cells, epithelial cells, keratinocytes, fibroblasts, fibrocytes and smooth muscle cells. IL-33 can be induced by immune stimuli. For instance, pro-inflammatory stimuli or pathogen recognition receptor (PRRs), especially TLRs, induce IL-33 expression in immune cells [14]. Similarly, in human keratinocytes IL-33 expression is constitutively weak but is strongly induced during inflammation. In contrast, although IL-33 is also constitutively expressed in mouse epithelial cells of various origins, expression is lost during acute inflammation. However, this is thought to be due to its release to perform its alarmin function.

IL-33 exhibits two diverse functions. IL-33 can function as a cytokine that binds to ST2L and regulates the immune response, epithelial repair and activates Th2 cells. IL-33 can also function as a nuclear factor involved in maintaining barrier function through gene regulation. However, once the barrier is breached, IL-33 is released and functions as an ‘alarmin’. The structure of IL-33 is vital to its multiple functions. IL-33 shares the IL-1 family C-terminal core tetrahedron structure, and similar to other IL-1 cytokine members, IL-1β and IL-18, IL-33 is translated without a signal sequence for secretion [15].

Under homeostatic conditions IL-33 predominantly localizes to the nucleus as it possesses a nuclear localization signal within its N terminus. Here it functions as a nuclear factor, binding directly to chromatin in the nucleus. Chromatin interaction is enabled by a conserved homeodomain-like helix-turn-helix motif located in the N-terminal domain. Nuclear IL-33 regulates gene expression in numerous ways. IL-33 binds to the nucleosome acidic patch in histone H2A-H2B dimers and regulates chromatin structure and by default, gene expression [16]. IL-33 also activates histone deacetylase-3 (HDAC3) activity, indicating a potential role for IL-33 in modulating epigenetic regulation. IL-33 may also bind to the transcriptional repressor, histone methyltranserase SUV39H1. Additionally, nuclear IL-33 has been reported to directly bind to NF-κB, suppressing its activity. IL-33 has been shown to alter expression of the p65 NF-κB subunit by binding to the p65 promoter [17].

IL-33 functions as a cytokine once it has been secreted into the extracellular environment. Similar to other IL-1 family members, it binds to its specific plasma membrane receptor, ST2L. Once IL-33 has bound to ST2L, the receptor undergoes a conformational change, recruiting the IL-1RAcP, thus bringing the two intracellular TIR domains together. Signalling occurs as previously described in section 1.2 (IL-1 family signalling). Similar to other IL-1 signalling pathways, IL-33 signal transduction may result in the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, p38 MAPK and JNKs. IL-33 may therefore also activate the transcription factor AP-1 independently of NF-κB activation. Indeed, although TRAF6 appears to be required for IL-33-mediated NF-B activation, IL-33-mediated ERK activation may be TRAF6-independent. This indicates a level of variation between the IL-33/ST2 pathway and other IL-1R signalling pathways.

In certain cells, such as mast cells, IL-33/ST2L signalling appear to be more complex as the IL-1RAcP has been shown to be complexed to c-Kit, a receptor tyrosine kinase (RTK). ST2L cross-activates c-Kit, and this RTK regulates IL-33 signalling in mast cells [18]. c-Kit is activated by its ligand, stem cell factor (SCF). As well as mast cells, c-Kit is also expressed by haemopoiectic stem cells, progenitor cells, innate lymphoid cells and neoplastic cells. It is unclear whether other RTKs associate with the IL-33/ST2L complex, and are involved in attenuating IL-33 responses.

Downstream of IL-33 signalling, activation of this pathway has been shown to have far reaching effects in many different cell types. The number of genes or proteins modified varies depending on the cell type being examined. In RAW 264.7 murine macrophages, IL-33 stimulation altered the expression of over 670 proteins. In CCD-18Co, a subepithelial myofibroblast cell line, IL-33 upregulated 700 transcripts and downregulated 650 transcripts [19]. In human umbilical vein endothelial cells (HUVECs), IL-33 regulated over 300 genes. Further research on the response of cells to stimulation with IL-33 alone and combined with other cytokines may provide an insight into the in vivo crosstalk which occurs during inflammation.

Given the potent immune reactions that occur following IL-33-induced ST2 signalling it is essential that this pathway is tightly regulated. Activation of ST2L by IL-33 results in activation of focal adhesion kinase (FAK) and glycogen synthase kinase-3β (GSK-3β). Activated GSK-3β binds to ST2L at Ser446 and phosphorylates it at Ser442. This results in the swift internalization of ST2L. Transmembrane emp24 domain- containing (TMED1) is a protein involved in the vesicular trafficking of proteins. TMED1 co-precipitates with ST2L [20]. Once internalized, ST2L is polyubiquitinated by the E3 ubiquitin ligase FBXL19, leading to proteasomal degradation of ST2L. IL-33 and ST2L signalling can also be regulated in other ways. Unlike other IL-1 family members, there is no known antagonistic ligand for ST2L. However, SIGIRR is an IL-1R family member that associates with ST2L. It is known to negatively regulate IL-1R-mediated immune responses and may interact with ST2L to inhibit IL-33/ST2L signalling. Evidence of this comes from SIGIRR-/- mice where IL-33 has been shown to induce a greater inflammatory response in the absence of SIGIRR [21]. The heterodimer IL-1RAcP that makes up the IL-33/ST2 signalling complex is constitutively expressed at low levels and does not appear to be subject to notable modulation.

References

1. Tominaga, S.-i., A putative protein of a growth specific cDNA from BALB/C-3T3 cells is highly similar to the extracellular
2. Yanagisawa, K., et al., Presence of a novel primary response gene ST2L, encoding a product highly similar to the interleukin 1 receptor type 1. FEBS Lett, 1993. 318(1): p. 83-7.
3. Tago, K., et al., Tissue distribution and subcellular localization of a variant form of the human ST2 gene product, ST2V. Biochem Biophys Res Commun, 2001. 285: p. 1377 – 1383.
4. Gachter, T., A.K. Werenskiold, and R. Klemenz, Transcription of the interleukin-1 receptor-related T1 gene is initiated at
5. Baba, Y., et al., GATA2 Is a Critical Transactivator for the Human IL1RL1/ST2 Promoter in Mast Cells/Basophils: OPPOSING ROLES FOR GATA2 and GATA1 IN HUMAN IL1RL1/ST2 GENE EXPRESSION. Journal of Biological Chemistry, 2012. 287(39): p. 32689-32696.
6. Schmieder, A., G. Multhoff, and J. Radons, Interleukin-33 acts as a pro-inflammatory cytokine and modulates its receptor gene expression in highly metastatic human pancreatic carcinoma cells. Cytokine, 2012. 60.
7. Bourgeois, E., et al., The pro-Th2 cytokine IL-33 directly interacts with invariant NKT and NK cells to induce IFN-gamma production. Eur J Immunol, 2009. 39(4): p. 1046-55.
8. Kuchler, A., et al., Nuclear interleukin-33 is generally expressed in resting endothelium but rapidly lost upon angiogenic or proinflammatory activation. Am J Pathol, 2008. 173: p. 1229 – 1242.
9. Cayrol, C. and J. Girard, The IL-1-like cytokine IL-33 is inactivated after maturation by caspase-1. Proc Natl Acad Sci USA, 2009. 106: p. 9021 – 9026.
10. Lefrancais, E., et al., IL-33 is processed into mature bioactive forms by neutrophil elastase and cathepsin G. Proc Natl Acad Sci U S A, 2012. 109(5): p. 1673-8.
11. Zhao, W.H. and Z.Q. Hu, Up-regulation of IL-33 expression in various types of murine cells by IL-3 and IL-4. Cytokine, 2012. 58(2): p. 267-73.
12. Zhao, W. and Z. Hu, The enigmatic processing and secretion of interleukin-33. Cell Mol Immunol, 2010. 7(4): p. 260-2.
13. Kalashnikova, A.A., et al., The role of the nucleosome acidic patch in modulating higher order chromatin structure. J R Soc Interface, 2013. 10(82): p. 6.
14. Ali, S., et al., The Dual Function Cytokine IL-33 Interacts with the Transcription Factor NF-κB To Dampen NF-κB–Stimulated Gene Transcription. The Journal of Immunology, 2011. 187(4): p. 1609-1616.
15. Drube, S., et al., The receptor tyrosine kinase c-Kit controls IL-33 receptor signaling in mast cells. Blood, 2010. 115(19): p. 3899-906.
16. Maywald, R.L., et al., IL-33 activates tumor stroma to promote intestinal polyposis. Proc Natl Acad Sci U S A, 2015. 112(19): p. 27.
17. Garlanda, C., H.J. Anders, and A. Mantovani, TIR8/SIGIRR: an IL-1R/TLR family member with regulatory functions in inflammation and T cell polarization. Trends Immunol, 2009. 30(9): p. 439-46.
18. Haraldsen, G., et al., Interleukin-33 – cytokine of dual function or novel alarmin? Trends Immunol, 2009. 30: p. 227 – 233.