Abstract
The present invention pertains to an N-terminally truncated interleukin (IL)-38 protein, or functional variants thereof, as well as to nucleic acids and vectors encoding the truncated IL-38 peptide and recombinant cells comprising these nucleic acids or vectors. The invention shows that IL-38 is N-terminally processed and that the truncated version of the cytokine acts as an antagonist of immune activation in macrophages. This indicates a use of the truncated cytokine in the treatment and prevention of autoimmune disorders. The invention further provides pharmaceutical compositions comprising the truncated IL-38 protein, and method for screening modulators of the function of truncated IL-38.
Claims
1. An isolated truncated IL-38 protein, or a functional variant thereof, wherein said truncated IL-38 protein is N-terminally truncated compared to the amino acid sequence according to SEQ ID NO: 1, and wherein said truncation comprises at least 10 adjoining amino acids between positions 1 to 30 of SEQ ID NO: 1.
2. The isolated truncated IL-38 protein according to claim 1, wherein said truncated IL-38 protein has 2-50 amino acids truncated at its N-terminus as compared with wild type IL-38 protein (SEQ ID NO: 1).
3. The isolated truncated IL38 protein according to claim 2, wherein said truncated IL-38 protein has 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids truncated at its N-terminus as compared to the protein shown in SEQ ID NO: 1.
4. The truncated IL-38 protein according to claim 1, having an N-terminus that is not identical to the first 100, 50, 30, 20, or 19 amino acids of SEQ ID NO: 1.
5. A nucleic acid comprising a sequence coding for a truncated IL-38 protein according to claim 1.
6. The nucleic acid according to claim 5 comprising a sequence that when expressed produces a polypeptide consisting of the truncated IL-38 protein, or a functional variant thereof, wherein said truncated IL-38 protein is N-terminally truncated compared to the amino acid sequence according to SEQ ID NO: 1, and wherein said truncation comprises at least 10 adjoining amino acids between positions 1 to 30 of SEQ ID NO: 1; and not a full length IL-38 protein according to SEQ ID NO: 1.
7. A vector comprising a nucleic acid according to claim 5.
8. The vector according to claim 7, wherein the expressible sequence is operable linked to a promoter.
9. A recombinant cell, comprising: a nucleic acid with a sequence coding for a truncated IL-38 protein, or a functional variant thereof, wherein said truncated IL-38 protein is N-terminally truncated compared to the amino acid sequence according to SEQ ID NO: 1, and wherein said truncation comprises at least 10 adjoining amino acids between positions 1 to 30 of SEQ ID NO: 1; or a vector according to claim 7.
10. A pharmaceutical composition comprising a truncated IL-38 protein according to claim 1, for use in medicine, preferably for use in the treatment or prevention of an immune- or inflammatory disease.
11. An in-vitro method for modulating the immune response of a cell, the method comprising contacting said cell with a truncated IL-38 protein, or a functional variant thereof, wherein said truncated IL-38 protein is N-terminally truncated compared to the amino acid sequence according to SEQ ID NO: 1, and wherein said truncation comprises at least 10 adjoining amino acids between positions 1 to 30 of SEQ ID NO: 1, or by expressing in said cell a nucleic acid according to claim 5.
12. The method according to claim 11, wherein modulating the immune response is an inhibition of JNK signaling, in particular the inhibition of IL-6 release and TH17 generation.
13. A method for screening for modulators of the activity of truncated IL-38, comprising the steps of a. Providing a cell, b. Contacting said cell with microbe-associated molecular pattern (MAMP), pathogen-associated molecular patterns (PAMP) or apoptotic cell supernatants (ACM), c. Further contacting said cell with a truncated IL-38 protein according to claim 1 and a candidate modulator, d. Determining INK activation in said cell, wherein an increase of INK activation in said cell compared to a control cell or reference value indicates that the candidate modulator is an antagonist of truncated IL-38, and a decrease of JNK activation compared to a control cell or reference indicates that the candidate modulator is an agonist of truncated IL-38.
14. The method according to claim 13, wherein said cell expresses on the cell surface a receptor of truncated IL-38, for example by ectopically expressing IL-1RAPL1 in said cell.
15. The method according to claim 13, wherein said JNK activation is determined by means of an AP-1 reporter construct.
16. A pharmaceutical composition comprising a nucleic acid according to claim 5, for use in medicine, preferably for use in the treatment or prevention of an immune or inflammatory disease.
17. A pharmaceutical composition comprising a vector according to claim 7, for use in medicine, preferably for use in the treatment or prevention of an immune or inflammatory disease.
18. A pharmaceutical composition comprising a recombinant cell according to claim 9, for use in medicine, preferably for use in the treatment or prevention of an immune or inflammatory disease.
Description
[0076] The present invention will now be further described in the following examples with reference to the accompanying figures and sequences, nevertheless, without being limited thereto. For the purposes of the present invention, all references as cited herein are incorporated by reference in their entireties. In the Figures:
[0077] FIG. 1: IL-38 is secreted from apoptotic cells. (A) A549 human lung cancer cells, MDA-231 breast cancer cells, human primary PBMCs and human primary neutrophils remained viable, were treated with TNF-alpha (20 ng/ml)/CHX (10 μM) to include apoptosis or were incubated at 60° C. for 30 min to induce necrosis. Respective supernatants of viable (VCM), apoptotic (ACM) or necrotic (NCM) cells were harvested and IL-38 levels were analyzed by ELISA. Data are means±SEM, n=5. (B) Secretion of IL-38 from apoptotic A549 cells was analyzed by ELISA at the times indicated. Data are means±SEM, n=5. *p<0.05, ANOVA with Bonferroni's correction.
[0078] FIG. 2: Apoptotic tumor cell-released IL-38 regulates cytokine production in macrophages. Human macrophages were stimulated for 24 h with (A) LPS (1 ng/ml) alone or in combination with recombinant human IL-38 (rhIL-38) long/ml or with (B) supernatants of apoptotic A549 cells (ACM) alone or in combination with recombinant human IL-38 (rhIL-38) 10 ng/ml. Cytokine production was measured using cytometric bead array, normalized results are shown. Data are means±SEM, n=5 (C) Human macrophages were stimulated with supernatants of viable (VCM) or apoptotic A549 cells (ACM), which were previously transfected with non-targeting siRNA (siCtrl), siRNA directed against IL-38 (siIL38) or an IL-38 overexpression vector (oeIL-38). Cytokine production was measured using cytometric bead array, normalized results are shown. Data are means±SEM, n=5 (D) Human macrophages were transfected with an AP1 reporter construct and luciferase activity was measured after 24 h stimulation with ACM from siCtrl and siIL-38 A549 cells. Background measurements obtained from mock-transfected cells were subtracted from each experimental value. Normalized results are shown. Data are means±SEM, n=5.*p<0.05, ANOVA with Bonferroni's correction.
[0079] FIG. 3: IL-38 binds to IL-1R6 and IL-1RAPL1. (A,B) Human macrophages were stimulated with LPS or ACM for 6 and 24 h. mRNA expression (A) and cell surface protein expression (B) of IL-1R6 and IL-1RAPL1 were measured by RT-qPCR and FACS respectively. Data are means±SEM, n=5. (C,D) Macrophages were controls (Ctrl) or incubated with 50 ng/ml IL-38 for 15 min on ice before staining with anti-IL-1R6 or anti-IL-1RAPL1 and their respective PE-conjugated secondary antibodies. Representative flow cytometry histograms (C) and statistical quantification of median PE intensity (D) are displayed. Data are means±SEM, n=5. (E) Binding kinetics of IL-38 to immobilized IL-1R6 and IL-1RAPL1. 96 well plates were coated with 0.5 μg of human IL-1R6 and IL-1RAPL1 extracellular domain-Fc chimeras and incubated with increasing amounts of human recombinant IL-38 as indicated. IL-38 binding to the extracellular domain of the receptors was detected using biotinylated monoclonal IL-38 antibody. Data are means±SEM, n=5.*p<0.05, ANOVA with Bonferroni's correction.
[0080] FIG. 4: Role of IL-1R6 and IL-1RAPL1 in cytokine production. Human macrophages were transfected with non-targeting siRNA (siCtrl) or siRNA directed against (A) IL-1RAPL1 (siIL1RAPL1) or (B) IL-1R6 (siIL1R6) and stimulated with VCM and ACM for 24 h. Cytokine production was measured using cytometric bead array, normalized results are shown. Data are means±SEM, n=5. *p<0.05, ANOVA with Bonferroni's correction.
[0081] FIG. 5: IL-38 regulates the Th17 response. Human T-cells were activated with anti-CD3/anti-CD28 beads, stained with eFluor 670 and stimulated with the supernatant of macrophages previously stimulated with ACM from control A549 cells (ACMshCtrl/MΦ) or IL-38 knock-down A549 cells (ACMshIL-38/MΦ). After 7 days (A) cytokine production and (B,C) cell proliferation was measured. (A) Cytokines were quantified using cytometric bead array, normalized results are shown. Data are means±SEM, n=10. T-cell proliferation was determined by following eFluor 670 dilution. (B) Statistical quantification of all proliferating T cells and (C) representative flow cytometry histograms are displayed. Data are means±SEM, n=10. *p<0.05, ANOVA with Bonferroni's correction.
[0082] FIG. 6: IL-38 is truncated at the N-Terminus. (A) The characteristic consensus motif of the IL-36 family in IL-38, which defines the putative cleavage site of this cytokine at the N-terminus, is displayed. (B) C-terminally myc-tagged IL-38 was over-expressed in A549 cells, which were then used for ACM production. After immunoprecipitating the over-expressed IL-38 using anti-myc coated beads, 2D gel electrophoresis was performed (isoelectric focusing at pH 4-7, followed by polyacrylamide gel separation), and a monoclocal anti-myc antibody was used to detect the immunoprecipitated IL-38 upon protein transfer onto nitrocellulose. (C) Coomasie-stained 2D gels were used for picking putative IL-38 spots, which were analyzed by mass spectrometry. Identified IL-38 N-terminal peptides for the different IL-38 spots are displayed.
[0083] FIG. 7: Full length and truncated IL-38 have opposite roles in cytokine production and bind to IL-1RAPL1. (A,C) Human macrophages were (A) untreated or (C) previously transfected with non-targeting siRNA (siCtrl) or siRNA directed against IL-1RAPL1 (siIL1RAPL1) and stimulated for 6 h with recombinant human IL-1β 50 ng/ml alone or in combination with different concentrations of recombinant human full length (IL-38aa1-152) or cleaved (IL-38aa20-152) IL38. After 24 h IL-6 concentration in the supernatants was measured using cytometric bead array, normalized results are shown. Data are means±SEM, n=7. (B) Binding kinetics of full length and cleaved IL-38 to immobilized IL1RAPL1. 96 well plates were coated with 0.5 μg of human IL-1RAPL1 extracellular domain-Fc chimeras and incubated with increasing amounts of human recombinant IL-38aa1-152 or IL-38aa20-152 as indicated. IL-38 binding to the extracellular domain of the receptors was detected using biotinylated monoclonal IL-38 antibody. Data are means±SEM, n=5.*p<0.05, ANOVA with Bonferroni's correction.
[0084] FIG. 8: IL-1RAPL1-induced signalling pathways regulated by IL-38 in HEK cells. HEK cells were co-transfected with an IL-1RAPL1 over-expression plasmid in combination with (A,D) AP1, (B,E) NFκB or (C) IL-6 reporter constructs. HEK cells transfected with the reporter constructs together with an empty plasmid instead the IL-1RAPL1 overexpression plasmid were used as controls (Mock). (A,B) HEK cells were stimulated with IL-1β (50 ng/ml) for 24 h and the AP1 or NFκB activity was measured. Normalized results are shown. Data are means±SEM, n=15. (C) IL-6 reporter constructs with point mutations in the indicated transcription binding sites were used. After transfection, cells were incubated for additional 24 h and IL-6 promoter-dependent luciferase activity was measured. Results are expressed as fold induction relative to Mock transfected cells. Data are means±SEM, n=10. (D,E) After transfection, fresh medium (Ctrl) or different concentrations of IL-38aa1-152 or IL-38aa20-152 were added and cells were incubated for additional 24 h. Results are expressed as fold induction relative to Mock transfected cells. Data are means±SEM, n=15. (F) IL1RAPL1 was overexpressed in HEK cells and after transfection fresh medium (Ctrl) or IL-38aa1-152 or IL-38aa20-152 (25 ng/ml) were added to the cells followed by incubation for additional 24 h. Intracellular staining of phosphorylated JNK and p38 was performed and measured by FACS. Results are expressed as fold induction relative to Mock transfected cells. Data are means±SEM, n=5. *p<0.05, ANOVA with Bonferroni's correction.
[0085] FIG. 9: IL-38 regulates AP1 activity in macrophages. Human macrophages were transfected with an empty vector, AP1, NFκB or IL-6 reporter constructs. After transfection, macrophages were stimulated for 24 h with IL-1β (50 ng/ml) alone or in combination with IL-38aa1-152 or IL-38aa20-152 (20 ng/ml). Luciferase activity was measured. Background measurements obtained from mock-transfected cells were subtracted from each experimental value. Normalized results are shown. Data are means±SEM, n=7. *p<0.05, ANOVA with Bonferroni's correction.
TABLE-US-00001 SEQ ID NO: 1 MCSLPMARYYIIKYADQKALYTRDGQLLVGDPVADNCCAEKICILPNRG LARTKVPIFLGIQGGSRCLACVETEEGPSLQLEDVNIEELYKGGEEATR FTFFQSSSGSAFRLEAAAWPGWFLCGPAEPQQPVQLTKESEPSARTKFY FEQSW SEQ ID NO: 2 LYTRDGQLLVGDPVADNCCAEKICILPNRGLARTKVPIFLGIQGGSRCL ACVETEEGPSLQLEDVNIEELYKGGEEATRFTFFQSSSGSAFRLEAAAW PGWFLCGPAEPQQPVQLTKESEPSARTKFYFEQSW
EXAMPLES
Example 1: IL-38 is Released from Apoptotic Cells
[0086] When performing an in-house ELISA to determine IL-38 levels produced by tumor cell lines, the inventors noticed that induction of apoptotic cell death markedly increased IL-38 secretion into the supernatant. Compared to the supernatant of viable A549 lung cancer or MDA.231 breast cancer cells (VCM), apoptotic cell supernatants (ACM), but not necrotic cell supernatants (NCM) contained approximately 10 fold higher levels of IL-38 (FIG. 1A). This was also the case for primary human neutrophils or PBMCs, although the increase of IL-38 release during apoptosis was not as strong (FIG. 1A). In order to analyze the kinetics of IL-38 secretion, the concentration of IL-38 in A549 supernatants was measured at different time points upon apoptosis induction. Enhanced IL-38 secretion was observed after 12 h following apoptosis induction (FIG. 1B), coinciding with the occurrence of apoptosis markers in A549 cells (data not shown).
Example 2: IL-38 Regulates Cytokine Production after ACM Stimulation
[0087] Apoptotic cell-derived mediators have the potential to modulate phagocyte responses, including cytokine production (26). The inventors analyzed the role of IL-38 in the production of a panel of cytokines that are produced upon macrophage activation by LPS or upon interaction with apoptotic cells (27). Of these, IL-6 and IL-8 production were regulated by IL-38. Addition of recombinant IL-38 to LPS-stimulated macrophages increased IL-6 and IL-8 production compared with LPS alone (FIG. 2A). Interestingly, when human macrophages were stimulated with ACM of A549 cells alone or in combination with recombinant human IL-38, the opposite effect was observed (FIG. 2B). IL-38 suppressed ACM-induced IL-6 and IL-8 secretion from macrophages. Since ACM already contained IL-38, the inventors wondered whether endogenous IL-38 affected cytokine macrophage cytokine production. To answer this question, IL-38 was over-expressed or knocked down in A549 cells before generating ACM. Indeed, stimulation of human macrophages with ACM produced from IL-38-overexpressing A549 cells resulted in reduced secretion of IL-6 and IL-8, whereas stimulation with ACM of IL-38 knock-down A549 cells yielded higher IL-6 and IL-8 concentrations (FIG. 2C). Among the prominent transcription factors that regulate cytokine production and are regulated by the IL-1 family are NFκB and AP1. As NFκB is blocked after interaction of macrophages with apoptotic cells (28), the inventors asked whether endogenous IL-38 regulated AP1 activation in response to ACM. When applying ACM of IL-38 knock-down A549 cells in comparison to control ACM to macrophage transfected with an AP1 luciferase reporter construct, the inventors noticed that ACM containing lower levels of IL-38 induced a more pronounced AP1 activation (FIG. 2D). In conclusion, endogenous IL-38 restricted inflammatory macrophage activation in response to apoptotic cell supernatants.
Example 3: IL-38 Antagonizes IL1RAPL1-Dependent Cytokine Production in Response in ACM
[0088] The inventors hypothesized that IL-38 inhibits ACM-induced cytokine production by acting as a receptor antagonist. Therefore, the inventors analyzed candidates of the IL-1 receptor family for their association with IL-38. It was shown that IL-38 binds to the IL-1R6 (19) and the inventors observed that the orphan receptor IL-1RAPL1 regulates cytokine production in macrophages after ACM stimulation (16). The inventors first determined the expression of IL1R6 and IL-1RAPL1 in macrophages was determined at mRNA level using qPCR (FIG. 3A) and at the level of cell surface availability by FACS (FIG. 3B) after ACM or LPS stimulation. IL1R6 expression was generally low (FIG. 3C) and was further down-regulated at the mRNA level after ACM or LPS stimulation, which was nevertheless not apparent at the cell surface expression level. Contrarily, IL-1RAPL1 expression was abundant (FIG. 3C) and was further induced both at the mRNA level as well as on the cell surface at 6 h following LPS and at 6 h and 24 h following ACM treatment (FIG. 3A, B). Moreover, IL-1RAPL1 expression at the cell surface was reduced after 24 h stimulation with LPS. These experiments suggested IL-1RAPL1 at least as an additional candidate for the action of IL-38. Next, the inventors analyzed whether IL-38 would bind to IL-1RAPL1 by performing both competition assays and receptor binding assays. For competition assays, human macrophages were incubated with recombinant human IL-38 before analyzing surface expression of IL-1R6 or IL1RAPL1. Based on the low level of IL-1R6 surface expression, it was difficult to see to observe differences in cell surface expression due to IL-38 pre-incubation (FIG. 3C, D). However, for IL-1RAPL1 the inventors observed that IL-38 competed with the antibody used for the FACS staining (FIG. 3C, D), indicating that IL-38 may bind to IL-1RAPL1. To validate these results, direct receptor binding assays were performed. Plates were coated with IL-1R6-Fc and IL-1RAPL1-Fc chimeras, different IL-38 concentrations were added to the wells and the bound IL-38 was visualized. As shown recently (19) IL-38 indeed bound to IL-1R6 (FIG. 3E). Moreover, IL-38 also bound to IL-1RAPL1 (FIG. 3E). As these results suggested that IL-38 might regulate cytokine production by binding to IL-1RAPL1, the inventors asked for the role of IL-1RAPL1 in ACM-induced cytokine production. Transient knock-down of IL-1R6 or IL-1RAPL1 was performed in human macrophages and IL-6 and IL-8 levels in macrophage culture supernatants were measured after ACM stimulation. IL-6 and IL-8 production after ACM stimulation were IL-1RAPL1 dependent (FIG. 4A), whereas IL-1R6 was not involved in cytokine production in the inventor's model (FIG. 4B).
Example 4: IL-38 Regulates Th17 Responses
[0089] Next the inventors asked for downstream consequences of IL-38-dependent suppression of cytokine production from macrophages by analyzing the effect of macrophages supernatants on T cell activation. The inventors isolated primary human T-cells, stimulated them with anti-CD3/antiCD28 beads and incubated them repeatedly with supernatants of macrophages previously stimulated with ACM and with ACM of IL-38 knock-down A549 cells. IL-10, IL-17 and IFN-γ levels were measured in the supernatants of the T-cells after seven days of culture. When macrophages were stimulated with ACM, their supernatants reduced IFN-γ and IL-10 production by T cells and slightly elevated IL-17 levels (FIG. 5A). Nevertheless, when macrophages were stimulated with ACM lacking IL-38, their supernatants strongly elevated IL-17 production by T cells and further decreased IL-10 concentrations (FIG. 5A). These effects were independent of differences in T cell proliferation. Treatment with ACM did not affect the number of proliferating T cells (FIG. 5B), although it affected the number of divisions pre dividing T cells, which might explain the reduced IFN-γ and IL-10 levels (FIG. 5C). However, there was no difference in T cell proliferation whether ACM contained IL-38 or not (FIG. 5B, C). These data show that IL-38 from apoptotic cells restricts the macrophage-dependent generation of Th17 cells and maintains IL-10 expression.
Example 5: IL-38 is N-Terminally Processed in Apoptotic Cells
[0090] Except for IL-1Ra all members of the IL-1 family are produced as precursors, which need to be cleaved at the N-terminus in order to reach full activity. Recently, according to the size of the N-terminal pro-domain, IL-38 was classified into the IL-36 subfamily (4, 19). IL-38, as the other members of this subfamily, possesses a consensus motif, which putatively determines the N-terminal cleavage site (FIG. 6A). In order to determine whether or not apoptosis induced IL-38 processing, C-terminally myc-tagged IL-38 was overexpressed in tumor cells and ACM was produced from these cells. After immunoprecipitating IL-38 2D gel electrophoresis was performed to visualize IL-38 isoforms. Two IL-38 isoforms were successfully identified in the gel, indicating that IL-38 is indeed processed during apoptosis (FIG. 6B). The two spots representing putative IL-38 isoforms were picked and analyzed by mass spectrometry (MS). In the spot with higher molecular weight, predicted as full length IL-38, two N-terminal peptides were found in the MS analysis, one from amino acid 9 to 18, and the second one from amino acid 24 to 41, whereas in the sample with lower molecular weight only the peptide from amino acid 24 to 41 was found (FIG. 6C). Thus, IL-38 is N-terminally processed in apoptotic cells.
Example 6: Full Length and Truncated IL-38 Exert Opposite Roles on the Regulation of Cytokine Production Through IL-1RAPL1
[0091] In order to determine whether full-length and truncated IL-38 have a different biological activity, IL-6 concentration in the supernatants of human macrophages stimulated with IL-1β, alone or in combination with different concentrations of the full-length (IL-38aa1-152) or truncated (IL-38aa20-152) IL-38, was determined. After IL-β stimulation, higher concentrations of IL-38aa1-152 (20 ng/ml, 10 mg/ml) significantly increased IL-6 production, whereas IL-38aa20-152 decreased IL-6 production even when applied at low concentration (FIG. 7A). Since IL-38 in ACM regulated IL-6 production by interacting with IL-1RAPL1, the inventors asked whether both IL-38 isoforms, which have opposite roles in cytokine production, bind to IL-1RAPL1. The inventors performed a receptor binding assay as explained above. Both IL-38 isoforms bound to IL-1RAPL1 in this assay (FIG. 7B). However, binding kinetics seemed to differ slightly. When considering that even though IL-38aa1-152 and IL38aa20-152 exert opposite roles on cytokine production, they are both able to bind to IL1RAPL1, another key point to analyze was whether or not the effects on IL-6 production were both IL-1RAPL1 dependent. To achieve this, a transient IL-1RAPL1 knock-down was performed in macrophages and IL-6 concentration in the supernatants was measured after stimulation with IL-1β alone or in combination with IL-38aa1-152 or IL-38aa20-152. IL-1RAPL1 knock-down in macrophages abrogated both, IL-6 induction by full-length 11-38 and IL-6 suppression by truncated IL-38 (FIG. 7C).
Example 7: IL-38 Regulates the IL-1RAPL1-Activated Pathway JNK/AP1
[0092] The inventors obtained evidence that IL-38 regulates AP.-1 in macrophages upon interaction with apoptotic cells (FIG. 2D). To analyze the signaling pathways that are affected by IL-38 in relation to its interaction with IL-1RAPL1, the inventors first utilized a receptor-overexpression model with HEK 293T cells. The cells were co-transfected with an overexpression construct for IL-1RAPL1 and AP1 or NFκB reporter constructs. HEK cells transfected with the reporter constructs but without over-expression of IL-1RAPL1 were used as control. First, to characterize the model IL-1RAPL1 over-expressing cells and control cells were stimulated with IL-1β, and AP1 (FIG. 8A) or NFκB (FIG. 8B) activity was measured. IL-1β was used as a low-affinity ligand for the orphan receptor IL-1RAPL1 (14). After IL-1β stimulation, a significant induction of NFκB but not AP1 activity was observed in control cells. Nevertheless, when IL-1RAPL1 was over-expressed an activation of AP1 as well as enhanced NFκB activity was observed. Thus, IL-1β alone induces NFκB activation in an IL1RAPL1-independent manner, but not AP1 activation, which required IL-1RAPL1. Interestingly, even without any stimulus, the presence of IL-1RAPL1 was sufficient to increase of AP1 and NFκB activities compared to control cells (FIG. 8A,B). IL-1RAPL1 therefore activates AP1, but not for NFκB, after IL-1 β stimulation in HEK cells, but induces AP1 and NFκB activation upon overexpression without addition of an exogenous ligand. To confirm this, IL-6 promoter constructs with or without point mutations in different transcription factor binding sites (AP1, NFκB, CREB and CEBPβ) were used. HEK cells were co-transfected with an IL1RAPL1 over-expression plasmid and IL-6 reporter constructs (29). Also in this set-up, over-expression of IL-1RAPL1 activated the IL-6 promoter compared with HEK control cells. This IL-6 promoter induction was abrogated when the AP1 and NFκB binding sites were mutated (FIG. 8C). This suggests the presence of an endogenous ligand for IL1RAPL1 that produced by HEK cells. Next the inventors asked whether IL-38aa1-152 and IL38aa20-152 were able to affect AP1 and NFκB activity downstream of IL-1RAPL1. NFκB promoter activity in this set-up was not regulated by IL-38 (FIG. 8D), but AP1 induction was negatively regulated by both IL-38 isoforms (FIG. 3E). Importantly, IL-38aa20-152 was able to regulate the AP1 induction at lower concentrations compared to the full-length protein. To analyze the signaling pathways leading to IL-38-dependent suppression of IL1RAPL-induced AP1 activity, intracellular staining of phosphorylated JNK and p38 was performed in IL-1RAPL1 over-expressing cells compared with control HEK cells. After IL1RAPL1 over-expression an induction in phosphorylated JNK but not p38 was observed. This induction was significantly reduced by IL-38aa20-152 but not by IL-38aa1-152, confirming the stronger regulatory role of truncated IL-38.
Example 8: IL-38 Regulates AP1 Activity in Macrophages
[0093] Next, the inventors transferred the inventor's data from the HEK model into the macrophage setting. Human macrophages were transfected with AP1 or NFκB reporter constructs and stimulated with IL-1β alone or in combination with IL-38aa20-152 or IL-38aa1-152. As in HEK cells, IL-38aa20-152 decreased AP1, but not NFκB activity in macrophages, whereas IL-38aa1-152 was ineffective (FIG. 9). Thus, only truncated IL-38 suppressed AP-1 activity in macrophages, which is in concordance with the inventor's finding that macrophages stimulated with apoptotic cell supernatants lacking IL-38 showed higher levels of AN activity (FIG. 2D). Finally the inventors approached the question, why IL-38aa1-152 increased IL-6 production after IL-1β stimulation of macrophages (FIG. 7A), whereas in the HEK cell model, IL-38aa1-152 did neither increase AN nor NFκB activation. To investigate this discrepancy macrophages were transfected with an IL-6 reporter construct and stimulated with IL-1β alone or in combination with both IL-38 isoforms. As expected, IL-38aa20-152 reduced IL-6 promoter activity, but IL-38aa1-152 did not affect IL-6 promoter induction at all (FIG. 9), suggesting that the IL-38aa1-152 mediated increase of IL-6 production was not transcriptionally regulated.
[0094] In conclusion, the present invention shows an N-terminally processed IL-38 which can be used in the clinic for limiting auto-inflammation in general or resulting, e.g., from defective interaction of macrophages with apoptotic cells.
REFERENCES
[0095] 1. Sims, J. E. & Smith, D. E. (2010) Nat Rev Immunol 10, 89-102. [0096] 2. Dinarello, C. A. (2009) Annu Rev Immunol 27, 519-50. [0097] 3. Dinarello, C. A. (2013) Seminars in immunology. [0098] 4. Garlanda, C., Dinarello, C. A. & Mantovani, A. (2013) Immunity 39, 1003-18. [0099] 5. Towne, J. E., Renshaw, B. R., Douangpanya, J., Lipsky, B. P., Shen, M., Gabel, C. A. & Sims, J. E. (2011) J Biol Chem 286, 42594-602. [0100] 6. Latz, E., Xiao, T. S. & Stutz, A. (2013) Nat Rev Immunol 13, 397-411. [0101] 7. Boraschi, D. & Tagliabue, A. (2013) Seminars in immunology. [0102] 8. Pavlowsky, A., Gianfelice, A., Pallotto, M., Zanchi, A., Vara, H., Khelfaoui, M., Valnegri, P., Rezai, X., Bassani, S., Brambilla, D., Kumpost, J., Blahos, J., Roux, M. J., Humeau, Y., Chelly, J., Passafaro, M., Giustetto, M., Billuart, P. & Sala, C. (2010) Current biology: CB 20, 103-15. [0103] 9. Gambino, F., Kneib, M., Pavlowsky, A., Skala, H., Heitz, S., Vitale, N., Poulain, B., Khelfaoui, M., Chelly, J., Billuart, P. & Humeau, Y. (2009) The European journal of neuroscience 30, 1476-86. [0104] 10. Jin, H., Gardner, R. J., Viswesvaraiah, R., Muntoni, F. & Roberts, R. G. (2000) European journal of human genetics: EJHG 8, 87-94. [0105] 11. Born, T. L., Smith, D. E., Garka, K. E., Renshaw, B. R., Bertles, J. S. & Sims, J. E. (2000) The Journal of biological chemistry 275, 29946-54. [0106] 12. Carrie, A., Jun, L., Bienvenu, T., Vinet, M. C., McDonell, N., Couvert, P., Zemni, R., Cardona, A., Van Buggenhout, G., Frints, S., Hamel, B., Moraine, C., Ropers, H. H., Strom, T., Howell, G. R., Whittaker, A., Ross, M. T., Kahn, A., Fryns, J. P., Beldjord, C., Marynen, P. & Chelly, J. (1999) Nature genetics 23, 25-31. [0107] 13. Khan, J. A., Brint, E. K., O'Neill, L. A. & Tong, L. (2004) The Journal of biological chemistry 279, 31664-70. [0108] 14. Pavlowsky, A., Zanchi, A., Pallotto, M., Giustetto, M., Chelly, J., Sala, C. & Billuart, P. (2010) Commun Integr Biol 3, 245-7. [0109] 15. Arthur, J. S. & Ley, S. C. (2013) Nat Rev Immunol 13, 679-92. [0110] 16. Ley, S., Weigert, A., Heriche, J. K., Mille-Baker, B., Janssen, R. A. & Brune, B. (2013) Immunobiology 218, 40-51. [0111] 17. Lin, H., Ho, A. S., Haley-Vicente, D., Zhang, J., Bernal-Fussell, J., Pace, A. M., Hansen, D., Schweighofer, K., Mize, N. K. & Ford, J. E. (2001) The Journal of biological chemistry 276, 20597-602. [0112] 18. Bensen, J. T., Dawson, P. A., Mychaleckyj, J. C. & Bowden, D. W. (2001) Journal of interferon & cytokine research: the official journal of the International Society for Interferon and Cytokine Research 21, 899-904. [0113] 19. van de Veerdonk, F. L., Stoeckman, A. K., Wu, G., Boeckermann, A. N., Azam, T., Netea, M. G., Joosten, L. A., van der Meer, J. W., Hao, R., Kalabokis, V. & Dinarello, C. A. (2012) Proc Natl Acad Sci USA 109, 3001-5. [0114] 20. Jung, M. Y., Kang, S. W., Kim, S. K., Kim, H. J., Yun, D. H., Yim, S. V., Hong, S. J. & Chung, J. H. (2010) Scandinavian journal of rheumatology 39, 190-6. [0115] 21. Guo, Z. S., Li, C., Lin, Z. M., Huang, J. X., Wei, Q. J., Wang, X. W., Xie, Y. Y., Liao, Z. T., Chao, S. Y. & Gu, J. R. (2010) International journal of immunogenetics 37, 33-7. [0116] 22. Chou, C. T., Timms, A. E., Wei, J. C., Tsai, W. C., Wordsworth, B. P. & Brown, M. A. (2006) Annals of the rheumatic diseases 65, 1106-9. [0117] 23. Monnet, D., Kadi, A., Izac, B., Lebrun, N., Letourneur, F., Zinovieva, E., Said-Nahal, R., Chiocchia, G. & Breban, M. (2012) Annals of the rheumatic diseases 71, 885-90. [0118] 24. Rahman, P., Sun, S., Peddle, L., Snelgrove, T., Melay, W., Greenwood, C. & Gladman, D. (2006) Arthritis and rheumatism 54, 2321-5. [0119] 25. Dehghan, A., et al. (2011) Circulation 123, 731-8. [0120] 26. Zitvogel, L., Kepp, 0. & Kroemer, G. (2010) Cell 140, 798-804. [0121] 27. Ley, S., Weigert, A., Weichand, B., Henke, N., Mille-Baker, B., Janssen, R. A. & Brune, B. (2013) Oncogene 32, 631-40. [0122] 28. Weigert, A., Tzieply, N., von Knethen, A., Johann, A. M., Schmidt, H., Geisslinger, G. & Brune, B. (2007) Mol Biol Cell 18, 3810-9. [0123] 29. Xiao, W., Hodge, D. R., Wang, L., Yang, X., Zhang, X. & Farrar, W. L. (2004) Prostate 61, 354-70.
