2026 年 51 巻 1 号 p. 1-9
Interleukin-1 receptor type 2 (IL-1R2) functions as a decoy receptor that suppresses IL-1-induced inflammatory signaling. Both membrane-bound IL-1R2 (WT IL-1R2) and its soluble form (sIL-1R2) bind interleukin-1α (IL-1α) at the cell surface or in the extracellular space, thereby inhibiting downstream signaling. However, the anti-inflammatory role of IL-1R2 varies depending on the cellular context and receptor structure. In this study, we generated two IL-1R2 deletion mutants—ΔTM, lacking the transmembrane domain, and ΔTMCP, lacking both the transmembrane and cytoplasmic domains—and compared their functions with those of WT IL-1R2 in HeLa cells. Western blotting, immunoprecipitation, and enzyme-linked immunosorbent assay were used to assess receptor expression, IL-1α binding, and IL-1β-induced interleukin-8 (IL-8) production, respectively. Both ΔTM and ΔTMCP were secreted more efficiently than WT IL-1R2. WT IL-1R2 exhibited weak intracellular interaction with IL-1α, whereas the deletion mutants showed minimal binding. WT IL-1R2 most effectively suppressed IL-1α extracellular release; however, ΔTM and ΔTMCP also reduced secretion. Notably, both deletion mutants suppressed IL-1β-induced IL-8 production more effectively than WT IL-1R2, indicating enhanced extracellular decoy activity. These findings demonstrate that structural modifications of IL-1R2 influence its function as a decoy receptor, and the enhanced inhibitory effects of the deletion mutants on IL-1 signaling provide new insight into the anti-inflammatory potential of soluble IL-1R2 in non-immune cells.
Key words: Interleukin-1, Interleukin-1 receptor type 2, decoy receptor, transmembrane, soluble interleukin-1 receptor type 2
Graphical Abstract
Molecules released from damaged or dying cells are collectively referred to as danger-associated molecular patterns (DAMP). Interleukin-1α (IL-1α) is a representative DAMP that plays a critical role in immune responses (Chen et al., 2007; Eigenbrod et al., 2008). IL-1α is synthesized in cells as a precursor IL-1α (pIL-1α), which is cleaved near its central region by proteases such as calpain and granzyme B. This cleavage yields the N-terminal propiece IL-1α (ppIL-1α) and the C-terminal mature IL-1α (mIL-1α) (Afonina et al., 2011, 2015). Among these, pIL-1α and mIL-1α are secreted extracellularly and induce inflammation by binding to specific receptors expressed on neighboring cells (Di and Shayakhmetov, 2016).
The receptor for IL-1α belongs to the IL-1 receptor family, which currently includes eleven members (Boraschi et al., 2018). IL-1 receptor type 1 (IL-1R1) and 2 (IL-1R2) are the prototypical receptors in this family. IL-1R1 is a type I membrane protein expressed on the cell surface. Upon binding to IL-1α, it associates with the accessory protein IL-1R3 (IL-1 receptor accessory protein; IL-1RAcP), which brings their respective Toll/IL-1 receptor (TIR) domains in close proximity, thereby initiating intracellular signaling (Weber et al., 2010). This signaling induces the production of proinflammatory cytokines, such as interleukin-6 (IL-6) and interleukin-8 (IL-8), via the activation of the NF-κB and MAPK pathways (Orjalo et al., 2009; Yano et al., 2008).
In contrast, IL-1R2 possesses only a short 29-amino-acid cytoplasmic region and lacks a TIR domain. Therefore, although it binds to IL-1α, it does not transmit signals and instead functions as a decoy receptor that suppresses inflammation (Colotta et al., 1993; Symons et al., 1995). Based on structural differences, IL-1R2 can be classified into four types: (i) membrane-bound (mIL-1R2), (ii) soluble secreted (ssIL-1R2), (iii) shed (shIL-1R2), and (iv) intracellular domain (IL-1R2ICD) (Garlanda et al., 2013; Schlüter et al., 2018; Zhang et al., 2024). mIL-1R2 contains a signal sequence that directs it to the ribosome during translation, after which it is transported to the plasma membrane via the ER-Golgi secretory pathway. As a type I membrane protein, IL-1R2 is retained (trapped) on the cell membrane through its transmembrane domain. Previous studies have indicated that membrane trapping is crucial for IL-1R2 to function effectively as a decoy receptor (Bethani et al., 2010; Peters et al., 2013; Westerfield and Barrera, 2020). Furthermore, IL-1R2 is cleaved near its juxtamembrane region by enzymes such as ADAM17 (also known as TACE), secretases, and aminopeptidases, producing a soluble form Interleukin-1 receptor type2 (sIL-1R2) that is released extracellularly (Afonina et al., 2011; Giai et al., 2016; Kuhn et al., 2007; Uchikawa et al., 2015). IL-1R2 also undergoes alternative splicing, generating multiple variants; in certain immune cells, such as B cells, sIL-1R2 is directly produced (Arend et al., 1994; Vambutas et al., 2009). In addition, it has been reported that IL-1R2 may function intracellularly as a transcriptional regulator (Mar et al., 2015).
The decoy function of IL-1R2 is thought to vary among cell types (Shimizu et al., 2015). In this study, we focused on HeLa cells to investigate how structural differences in IL-1R2 regulate cellular responses to IL-1β. Although the decoy function of membrane-bound IL-1R2 has been extensively studied in immune cells, such as macrophages, the role of soluble IL-1R2, particularly in non-immune cells, remains poorly understood. Moreover, several isoforms of soluble IL-1R2 have been reported, but their relative contributions to intracellular IL-1α retention and extracellular IL-1β neutralization have not been clearly delineated. Using deletion mutants of IL-1R2 lacking the transmembrane and cytoplasmic domains, this study aimed to dissect the domain-specific functions of IL-1R2 and provide novel insights into the anti-inflammatory role of soluble IL-1R2 in epithelial-like non-immune cells.
HeLa cells used in this study were procured from the RIKEN BioResource Research Center (BRC; Tsukuba, Japan). The identity of the cell line was confirmed through short tandem repeat (STR) profiling conducted by the supplier, which was consistent with a publicly available reference profile. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and maintained at 37°C in a 5% CO2 atmosphere. For the assays, 5 × 104 cells were seeded in each well of a 48-well plate. For western blotting (WB), cells were seeded at a density of 1 × 105 cells per well. All cell lines were routinely tested and confirmed to be free from Mycoplasma contamination using MycoStrip (InvivoGen, San Diego, CA, USA).
Expression plasmids and transfectionTo express IL-1α, a plasmid encoding full-length pIL-1α cDNA was inserted into the pcDNA3.1 vector, designated as pcDNA-pIL-1α (IL-1α) (Fig. 1C). For the expression of IL-1R2, a pCMV-SPORT6 vector containing wild-type (WT) IL-1R2 obtained from RIKEN BRC was used for transfection. This plasmid served as a template to generate two mutants via site-directed mutagenesis using the QuikChange site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA, USA): Δtransmembrane IL-1R2 (ΔTM), which lacks the 26-amino-acid transmembrane domain, and Δtransmembrane and cytoplasmic IL-1R2 (ΔTMCP), which lacks both the transmembrane and cytoplasmic domains (Fig. 1C). Transfection was performed using polyethyleneimine (PEI; 1 mg/mL, Thermo Fisher Scientific, Tokyo, Japan). The plasmids were mixed with 25 μL OPTI-MEM (Thermo Fisher Scientific) and 0.5 μL PEI, incubated at room temperature for 15 min, and subsequently added to the cells for transfection. Following an 18-h incubation period, the medium was replaced with fresh DMEM for culture.
Establishment of IL-1R2-deficient HeLa cells and expression of IL-1α and IL-1R2 variants
(A) Western blotting confirming IL-1R2 deletion in the R2-6 clone. Parental HeLa cells showed one or two IL-1R2 bands (55 and/or 46 kDa), which were absent in R2-6 cells. GAPDH served as a loading control. (B) Sanger sequence verification of the CRISPR-targeted IL-1R2 locus. Top: schematic representation of the human IL-1R2 gene, showing exons 1–5 (black boxes, E1–E5) and intervening introns (lines). The guide RNA used for CRISPR/Cas9-mediated knockout targets exon 3 of IL-1R2 (transcript ENST00000332549.8, MANE Select) on chromosome 2 (GRCh38). Middle and bottom: partial wild-type (WT) coding sequence and representative inferred mutant alleles from the R2-6 clone. In all sequences, the CRISPR guide RNA target is boxed and the PAM is underlined. Inserted nucleotides are shown in red and deletions are indicated by hyphens. Both representative alleles introduce frameshift mutations that generate premature stop codons downstream of the cut site (for Allele 2, the stop codon appears 10 amino acids after the region shown), consistent with the absence of full-length IL-1R2 protein in R2-6 cells. (C) Cartoon representation of IL-1α and IL-1R2 constructs. WT IL-1R2 retains extracellular (ECD), transmembrane (TM), and cytoplasmic (CP) domains. ΔTM lacks TM, and ΔTMCP lacks both TM and CP. ΔTM lacks the transmembrane region (amino acids 344–369), whereas ΔTMCP lacks both the transmembrane and cytoplasmic regions (amino acids 344–398). (D) Western blotting confirming expression of IL-1α (~35 kDa) and IL-1R2 WT, ΔTM, and ΔTMCP (~55, 48, and 40 kDa, respectively). GAPDH served as a loading control.
HeLa cells were derived from the authenticated RIKEN HeLa cell line (RCB0007). The IL-1R2 knockout cell line, designated R2-6, was developed using all-in-one vectors containing CRISPR/Cas9 guide RNA (gRNA) specific to IL-1R2 (Vector Builder, Yokohama, Japan). Transfection was conducted for 48–72 h using PEI. Post-transfection, the cells were cultured in the presence of puromycin (400 ng/mL) for two days and subsequently cloned through limiting dilution. Chromosomal DNA was extracted using a DNA extraction kit (FAVORGEN, Ping Tung, Taiwan) and subjected to DNA sequencing (FASMAC, Atsugi, Japan).
Western blotting and immunoprecipitation (IP)Transfected cells were washed with phosphate-buffered saline (PBS) and collected in lysis buffer composed of 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.5% Triton X-100. The cell lysates were centrifuged to eliminate debris. The resulting supernatants were combined with SDS sample buffer, heated at 95°C for 5 min, and analyzed via 12% SDS-PAGE. Proteins were subsequently transferred onto Immobilon transfer membranes (Merck, Darmstadt, Germany) and blocked with 1% bovine serum albumin (BSA) in PBS containing 0.1% Tween-20 (1% BSA–PBST).The primary antibodies used were rabbit anti-human IL-1α (1:1000; 16765-1-AP, Proteintech, Rosemont, IL, USA), mouse anti-human IL-1R2 (G-5; 1:500; sc-376247, Santa Cruz Biotechnology, Dallas, TX, USA), and rabbit anti-human GAPDH (FL-335; 1:10000; sc-25778, Santa Cruz Biotechnology). The secondary antibodies used were horseradish peroxidase-conjugated goat anti-mouse IgG and goat anti-rabbit IgG (AB_2338443, AB_2337910; 1:5000; 115-001-003, 111-001-003, Jackson ImmunoResearch, West Grove, PA, USA). For immunoprecipitation (IP), 450 μL of both the culture supernatant and cell lysate were collected from the transfectants, with 50 μL reserved as input for direct Western blot analysis. The remaining 400 μL was incubated with 500 ng of anti-IL-1α antibody at 4°C for 24 h with rotation. Subsequently, 30 μL of Protein G SepharoseTM 4 Fast Flow (Cytiva, Tokyo, Japan) was added and incubated at 4°C for 1 h. The resin was washed three times with PBS or lysis buffer and used as an IP sample.
Enzyme-linked immunosorbent assay (ELISA)To assess the extracellular secretion ratio of each IL-1R2 variant, HeLa cells were transfected with WT IL-1R2 or ΔTM and ΔTMCP variants, and cultured for 18 h. Following a 6-h media replacement, 300 μL of culture supernatant and cell lysate were collected and centrifuged to eliminate debris. IL-1R2 levels were quantified using a Human IL-1R2 ELISA Kit (Sino Biological, Beijing, China), and the extracellular secretion ratio was calculated as follows: [IL-1R2 in the supernatant/(IL-1R2 in the supernatant + lysate)] × 100 (%). To evaluate IL-1α secretion efficiency, the cells were co-transfected with IL-1α and either pcDNA, WT IL-1R2, ΔTM, or ΔTMCP vectors. After a 6-h media replacement, 300 μL of culture supernatant and cell lysate were collected. IL-1α levels were measured using the ELISA MAX Deluxe Set Human IL-1α (BioLegend, San Diego, CA, USA), and the secretion efficiency was calculated using the same formula described above. For IL-8 production in response to IL-1β, HeLa and R2-6 cells were transfected with pcDNA, WT IL-1R2, ΔTM, or ΔTMCP, followed by stimulation with recombinant IL-1β (50 pg/mL) for 6 h. Supernatants (300 μL) were collected and analyzed using the ELISA MAX Deluxe Set Human IL-8 (BioLegend). To investigate the mechanism of IL-1β response suppression, culture supernatants from HeLa cells transfected with the IL-1R2 variants or pcDNA were preincubated with recombinant IL-1β (50 pg/mL) for 1 h at 4°C and then applied to freshly seeded HeLa cells. After 6 h, the supernatants (300 μL) were collected and IL-8 levels were measured as described above.
StatisticsNormality was assessed using statistical software prior to hypothesis testing. Data shown in Fig. 2A (n = 7) and Fig. 2C (n = 8, vs. pcDNA), Fig. 2C (n = 8, vs. WT IL-1R2), Fig. 3A (n = 4, vs. WT IL-1R2), Fig. 3C (n = 5, vs. WT IL-1R2) followed a normal distribution and were analyzed using the parametric Student’s t-test. Data shown in Fig. 3A (n = 4, vs. pcDNA) and Fig. 3B (n = 7, vs. pcDNA), Fig. 3C (n = 5, vs. pcDNA), Fig. 3B (n = 7, vs. WT IL-1R2) did not follow a normal distribution and were analyzed using the nonparametric Mann–Whitney U test. Results are presented as the mean ± SD. Statistical significance was defined as p<0.05. All analyses were performed using R version 4.0.1 (R Core Team, Vienna, Austria; https://www.R-project.org/).
Secretion of IL-1R2 variants and suppression of IL-1α release
(A) Extracellular secretion efficiencies of IL-1R2 variants in HeLa cells measured by ELISA. ΔTM and ΔTMCP were secreted at significantly higher ratios than WT IL-1R2. Secretion efficiency was calculated as follows: [supernatant/(supernatant + lysate)] × 100 (%). Data are presented as means ± SD of seven independent experiments (n = 7). **p<0.01 vs. WT IL-1R2. (B) Co-immunoprecipitation showing interaction between IL-1α and IL-1R2 variants in HeLa cells. IL-1α was immunoprecipitated from cell lysates and culture supernatants using an anti-IL-1α antibody, and co-precipitated IL-1R2 variants were detected by Western blotting. Input panels show IL-1α and IL-1R2 in cell lysates and supernatants prior to IP. IL-1α was readily detected in lysate inputs but was below the detection limit in supernatant inputs, consistent with its low secretion ratio (~5%) measured by ELISA. A faint IL-1R2 band was detected predominantly in the lysate of WT-transfected cells, whereas ΔTM and ΔTMCP were more enriched in the supernatant, consistent with their higher secretion efficiencies. Ig heavy/light chains are indicated by arrows, and IL-1R2 is marked with a distinct arrowhead. The immunoglobulin heavy chain (~50 kDa) derived from the IP antibody is strongly detected, whereas the light chain (~25 kDa) signal is relatively weak. This is likely due to the higher detection sensitivity of the heavy chain and the short exposure time used, reflecting high IP efficiency for the target protein was high under these conditions. (C) IL-1α secretion was reduced by all IL-1R2 variants compared with control in HeLa cells. Secretion efficiency was calculated as follows: [supernatant/(supernatant + lysate)] × 100 (%). Data are presented as means ± SD of eight independent experiments (n = 8). *p<0.05, **p<0.01, vs. pcDNA control. n.s., not significant vs. WT IL-1R2.
Inhibitory effects of IL-1R2 variants on IL-1β-induced IL-8 production
(A–B) ELISA of IL-8 production in parental HeLa (A) or R2-6 (B) cells expressing IL-1R2 variants following IL-1β stimulation. ΔTM and ΔTMCP suppressed IL-8 levels more strongly than WT IL-1R2. Data are presented as mean ± SD of independent experiments (n = 4 for A and n = 7 for B). *p<0.05, **p<0.01, vs. pcDNA control; #p<0.05 vs. WT IL-1R2. (C) Neutralization assay using conditioned media from IL-1R2–expressing HeLa cells. Membrane localization contributed to WT IL-1R2 function but soluble forms (ΔTM, ΔTMCP) exhibited superior extracellular neutralization. Data are presented as the mean ± SD of five independent experiments (n = 5). **p<0.01 vs. pcDNA control; ##p<0.0001 vs. WT IL-1R2.
Using CRISPR/Cas9-mediated gene editing and subsequent revalidation via STR analysis, we first established a HeLa cell-derived IL-1R2 knockout cell line, designated R2-6. IL-1R2 deficiency was confirmed by WB analysis (Fig. 1A). In parental HeLa cells, IL-1R2 (55, 46 kDa) was observed mainly as a single band or as a closely spaced doublet, neither of which was detected in R2-6 cells. Sequence analysis indicated that the gRNA target region was mutated (Fig. 1B).
The expression of IL-1α, WT IL-1R2, ΔTM, and ΔTMCP in the transfected cells was verified by WB analysis (Fig. 1D). IL-1α was observed at approximately 35 kDa, whereas IL-1R2 WT, ΔTM, and ΔTMCP were detected at approximately 55, 48, and 40 kDa, respectively. Although the mobility differences between WT and the deletion mutants were not fully resolved in all gels, the observed sizes were consistent with the predicted molecular weights based on their truncation patterns. In overexpressing cells, WT IL-1R2 frequently appeared as a more clearly resolved doublet, whereas ΔTM showed a less distinct separation and ΔTMCP was predominantly observed as a single band, likely reflecting differences in post-translational processing between the full-length and truncated forms.
Extracellular secretion of IL-1R2 variants and inhibitory effect on IL-1α secretionThe extracellular secretion efficiencies of the three IL-1R2 variants were assessed in HeLa cells using an IL-1R2-specific ELISA. The secretion ratios were 14.16 ± 5.49% for WT IL-1R2, 76.20 ± 4.08% for ΔTM, and 66.33 ± 6.33% for ΔTMCP. These results indicate that both ΔTM and ΔTMCP variants were secreted with greater efficiency than WT IL-1R2 (Fig. 2A).
The interaction between IL-1α and each IL-1R2 variant was evaluated in HeLa cells using a co-immunoprecipitation assay (Fig. 2B). In the input samples, IL-1R2 from WT-transfected cells was detected mainly in the cell lysate, whereas ΔTM and ΔTMCP were more abundant in the culture supernatant, consistent with their higher secretion efficiencies. IL-1α was readily detected in the cell lysate inputs but was barely detectable in supernatant inputs under our Western blot conditions, indicating that the small secreted fraction of IL-1α (approximately 5% by ELISA; Fig. 2C) lies below the detection limit of this assay. After immunoprecipitation with an anti-IL-1α antibody, a faint IL-1R2 band was detected in the cell lysate from WT IL-1R2-transfected cells, whereas no clear IL-1R2 bands were observed for ΔTM and ΔTMCP in either lysates or supernatants. The relatively weak light-chain signal compared with the heavy chain likely reflects the higher detection sensitivity of the heavy chain and the shorter exposure time used owing to the high IP efficiency for the target protein.
These findings suggest that WT IL-1R2 forms weak but detectable intracellular complexes with IL-1α, whereas ΔTM and ΔTMCP do not form stable complexes that are readily detectable by IP–WB under these conditions, despite their comparable functional inhibition of IL-1α secretion. Consequently, we evaluated the impact of each IL-1R2 variant on IL-1α secretion. In HeLa cells co-transfected with IL-1α and pcDNA, the secretion efficiency of IL-1α was 9.25 ± 2.88%. In contrast, co-transfection with WT IL-1R2 reduced the secretion efficiency to 4.32 ± 1.52% (Fig. 2C). Co-transfection with ΔTM and ΔTMCP also resulted in decreased IL-1α secretion, with efficiencies of 5.81 ± 1.51% and 5.85 ± 1.47%, respectively. These results demonstrate that all IL-1R2 variants suppressed IL-1α secretion. Although the secretion efficiencies in ΔTM- and ΔTMCP- expressing cells tended to be lower than in WT IL-1R2-expressing cells, these differences did not reach statistical significance. Notably, while ELISA could detect the ~5–10% of IL-1α present in the culture supernatant, this low secreted fraction was below the detection limit of our WB conditions, which explains the absence of visible IL-1α bands in the supernatant lanes. Although immunoprecipitation followed by western blotting (IP–WB) revealed minimal interactions between IL-1α and the mutants, ΔTM and ΔTMCP were comparable to WT IL-1R2 in their ability to suppress IL-1α release.
Effects of IL-1R2 variants on IL-1β-induced IL-8 productionSubsequently, we investigated the effects of each IL-1R2 variant on IL-1β-induced IL-8 production. In HeLa cells transfected with pcDNA, the IL-8 concentration reached 109.58 ± 18.01 pg/mL, whereas wild-type (WT) IL-1R2 reduced this concentration to 68.91 ± 10.05 pg/mL. Notably, the ΔTM and ΔTMCP variants further suppressed IL-8 production to 51.76 ± 4.98 pg/mL and 46.27 ± 6.89 pg/mL, respectively (Fig. 3A). Conversely, in R2-6 cells transfected with pcDNA, the IL-8 concentration was 352.91 ± 52.88 pg/mL, whereas the WT IL-1R2 reduced this value to 70.62 ± 22.23 pg/mL. Remarkably, ΔTM and ΔTMCP variants demonstrated even greater efficacy in suppressing IL-8 production, reducing it to 33.32 ± 19.92 pg/mL and 33.48 ± 17.41 pg/mL, respectively (Fig. 3B). In both parental HeLa and R2-6 cells, ΔTM and ΔTMCP significantly reduced IL-1β-induced IL-8 production compared with WT IL-1R2.
To elucidate the mechanism by which IL-1R2 variants attenuate IL-1β-induced IL-8 production, we preincubated the culture supernatants from HeLa cells transfected with each IL-1R2 variant or pcDNA with IL-1β and subsequently applied them to freshly seeded new HeLa cell cultures. In the pcDNA group, IL-8 production was measured at 261.37 ± 32.14 pg/mL, whereas WT IL-1R2 reduced IL-8 levels to 144.73 ± 23.33 pg/mL. In contrast, the ΔTM and ΔTMCP variants further reduced IL-8 levels to 48.00 ± 9.78 pg/mL and 39.42 ± 13.69 pg/mL, respectively. However, the inhibitory effect of WT IL-1R2 in this assay was less pronounced than that in the direct transfection experiment (Fig. 2B), indicating a 35.36 ± 9.56% reduction (Fig. 3C). These findings suggest that although all IL-1R2 variants suppress IL-1β responsiveness, ΔTM and ΔTMCP exert stronger extracellular neutralizing effects than WT IL-1R2, while membrane localization remains particularly critical for the function of WT IL-1R2.
This study investigated the functional differences between WT IL-1R2 and its deletion mutants lacking the transmembrane or cytoplasmic domains, based on previous reports indicating that membrane trapping is essential for IL-1R2 decoy activity (Bethani et al., 2010; Peters et al., 2013; Westerfield and Barrera, 2020). The ΔTM and ΔTMCP mutants were secreted more efficiently than WT IL-1R2, confirming that the transmembrane domain anchors IL-1R2 to the plasma membrane. WB and IP analyses revealed that WT IL-1R2 weakly interacted with IL-1α intracellularly, whereas ΔTM and ΔTMCP showed minimal binding under our experimental conditions. Because IL-1α interacts with the extracellular domain of IL-1R2 and was barely detectable in the culture supernatant by Western blotting, these data suggest that WT IL-1R2 can form transient intracellular complexes with IL-1α, while the deletion mutants either interact only transiently or fail to form complexes that are stable enough to be detected by IP–WB.
WT IL-1R2 most effectively suppressed IL-1α secretion, indicating that membrane localization facilitates intracellular retention of IL-1α. pIL-1α is known to localize to the cell surface in monocytic cells (Conlon et al., 1987; Kurt-Jones et al., 1985; Matsushima et al., 1986), and IL-1R2 has been implicated in this process (Chan et al., 2020). Neumann et al. showed that blocking IL-1R2 shedding reduces responsiveness to extracellular IL-1, highlighting the importance of membrane-associated IL-1R2 (Neumann et al., 2000). Thus, IL-1R2 likely retains pIL-1α within cells, functioning as an intracellular decoy receptor.
Our immunofluorescence data (unpublished) suggested cytoplasmic distribution of IL-1R2, consistent with its role in intracellular IL-1α regulation. Although ΔTM and ΔTMCP also reduced IL-1α secretion, their weak interaction with IL-1α implies either transient or indirect effects, such as modulation of secretory pathways. It is unlikely that the apparent suppression results from undetected IL-1R2–IL-1α complexes, given the limited IP–WB detection.
Interestingly, ΔTM and ΔTMCP more effectively inhibited IL-1β-induced IL-8 production than WT IL-1R2, possibly due to enhanced extracellular decoy activity. In IL-1R2-deficient HeLa cells (R2-6), IL-8 levels were elevated overall but exhibited similar suppression patterns upon reintroduction of WT and mutant IL-1R2, confirming that both full-length and soluble forms retain intrinsic anti-inflammatory activity. Notably, we did not observe a consistent functional difference between ΔTM and ΔTMCP in any of our assays, suggesting that, at least in the context of short-term IL-1β signaling in HeLa cells, the cytoplasmic domain is not essential for the decoy activity of IL-1R2. Rather, the enhanced anti-inflammatory effects of these mutants are likely driven primarily by their increased secretion and the generation of soluble IL-1R2.
Few studies have examined IL-1R2 decoy functions in non-immune cells (Re et al., 1996; Schlüter et al., 2018; Shimizu et al., 2015). Our results extend this understanding by demonstrating that soluble IL-1R2 can suppress IL-1 signaling in epithelial-like cells. sIL-1R2, generated by ADAM17-mediated shedding (Black et al., 1997; Uchikawa et al., 2015), may also produce an intracellular domain (ICD) through γ-secretase cleavage (Kopan and Ilagan, 2004; Kuhn et al., 2007). The ICD reportedly interacts with transcription factors such as c-Fos to regulate epithelial gene expression (Mar et al., 2015), possibly contributing to tissue repair and inflammation resolution. Indeed, ADAM17-deficient mice display severe inflammation (Peschon et al., 1998), supporting this coordinated regulatory mechanism.
Elevated sIL-1R2 levels have been reported in rheumatoid arthritis (RA) patients (Arend et al., 1994; Jouvenne et al., 1998; Lin et al., 2012), and IL-1R2 knockout mice show exacerbated macrophage activation and arthritis symptoms (Iwakura, 2002; Shimizu et al., 2015). Although some aspects of IL-1R2 biology remain unclear, our findings indicate that soluble IL-1R2 binds and neutralizes IL-1 in epithelial cells, suggesting its potential as a therapeutic target for inflammatory diseases such as RA.
This work was supported by JSPS KAKENHI (Grant Number 22K17027, 25K24079), the Sato Fund (SATO-2023-5, SATO-2024-5, SATO-2025-5), the Uemura Fund, and the Dental Research Center at Nihon University School of Dentistry (DRC(A)-2023-3, DRC(A)-2024-3, DRC(A)-2025-3, DRC(B)-2024-5), and the Japanese Dental Science Federation (JDSF-DSP1-2025-120-1).
Conflict of Interest StatementThe authors declare that there are no conflicts of interest.
Data Availability StatementAll data generated or analyzed during this study are included in this published article and its supplementary materials.
Author Contribution StatementYH, MA, and SI conceived and supervised the study. IK, JI, YY, MY, YA, and MT performed the experiments and analyzed the data. All authors contributed to data interpretation and manuscript preparation. All authors have read and approved the final version of the manuscript.
Ethics Approval and Consent to ParticipateNot applicable.
Patient Consent for PublicationNot applicable.
Interleukin-1
IL-6Interleukin-6
IL-8Interleukin-8
IL-1R1Interleukin-1 receptor type 1
IL-1R2Interleukin-1 receptor type 2
IL-1αInterleukin-1 alpha
IL-1βInterleukin-1 beta
TIRToll/IL-1 receptor
sIL-1R2soluble form Interleukin-1 receptor type2
WTwild-type
KOknockout
PEIpolyethyleneimine
IPImmunoprecipitation
WBWestern blotting
IP-WBImmunoprecipitation followed by western blotting
ELISAenzyme-linked immuno-sorbent assay
DAMPdanger-associated molecular patterns
pIL-1αprecursor IL-1α
ppIL-1αpropiece IL-1α
mIL-1αmature IL-1α
ΔTMΔtransmembrane IL-1R2
ΔTMCPΔtransmembrane and cytoplasmic IL-1R2
STRshort tandem repeat
gRNAguide RNA
PBSphosphate-buffered saline
1% BSA-PBST1% bovine serum albumin (BSA) in PBS containing 0.1% Tween-20
RIPregulated intramembrane proteolysis
ICDintracellular domain
