Role of caspase-1 in nuclear translocation of IL-37, release of the cytokine, and IL-37 inhibition of innate immune responses

Abstract

IL-37 is a fundamental inhibitor of innate immunity. Human IL-37 has a caspase-1 cleavage site and translocates to the nucleus upon LPS stimulation. Here, we investigated whether caspase-1 processing affects IL-37-mediated suppression of LPS-induced cytokines and the release from cells by analyzing a caspase-1 cleavage site mutant IL-37 (IL-37D20A). Nuclear translocation of IL-37D20A is significantly impaired compared with WT IL-37 in transfected cells. LPS-induced IL-6 was decreased in cells expressing WT IL-37 but not IL-37D20A. The function of IL-37 in transfected bone marrow-derived macrophages is nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome-dependent, because IL-37 transfection in apoptosis-associated speck-like protein containing a carboxyl-terminal caspase recruitment domain- and NLRP3-deficient cells does not reduce levels of IL-6 and IL-1β upon LPS stimulation. IL-37-expressing macrophages release both precursor and mature IL-37, but only the externalization of mature IL-37 was dependent on ATP. Precursor and mature IL-37 was also secreted from human dendritic cells and peripheral blood mononuclear cells. To determine whether IL-37 is active in the extracellular compartment, we pretreated IL-37 transgenic mice with IL-37-neutralizing antibodies before LPS challenge. In IL-37-expressing mice, neutralizing IL-37 antibodies reversed the suppression of LPS-induced serum IL-6. In contrast, the addition of neutralizing antibody did not reverse suppression of LPS-induced IL-6 in mouse macrophages transfected with IL-37. Although caspase-1 is required for nuclear translocation of intracellular IL-37 and for secretion of mature IL-37, the release of the IL-37 precursor is independent of caspase-1 activation. IL-37 now emerges as a dual-function cytokine with intra- and extracellular properties for suppressing innate inflammation.

Fig. 1.

Mutation of caspase-1 cleavage site reduces processing of IL-37 after LPS stimulation. Single-cell clones of stable RAW transfectants expressing either IL-37 or IL-37D20A alone (A) or as a fusion protein with an N-terminal CFP tag (B) were challenged for 18 h with LPS (100 ng/mL). Cell lysates (total of 50 μg of protein per lane) were subjected to Western blotting using a murine mAb against human IL-37. A representative Western blot of four experiments is shown. mat, mature.

Fig. 2.

Nuclear translocation and anti-inflammatory function of IL-37 compared with IL-37D20A. (A) Cytoplasmic and nuclear extracts from RAW cells expressing IL-37 and IL-37D20A without or with 18 h of LPS stimulation (100 ng/mL) were subjected to Western blotting with monoclonal mouse anti-human IL-37. IL-37 in the nuclear extract is the mature form, which is running at a slightly higher molecular weight on the gel due to nucleic acid contamination in the nuclear protein sample. (B) Subcellular distribution of IL-37 and IL-37D20A as a fusion protein with a carboxyl-terminal tag 18 h after LPS stimulation (100 ng/mL) in transfected RAW cells. Red indicates nuclear staining, and green indicates IL-37 staining. One of three representative experiments performed is shown (Magnification: 100×). (C) Two single-cell clones of stable RAW transfectants expressing IL-37D20A (1, 2), one clone expressing WT IL-37, and one mock-transfected clone were stimulated with LPS (100 ng/mL) for 18 h. Mean ± SEM. IL-6 levels in supernatants of stimulated cells of four independent experiments are shown. ***P < 0.001 for mock-transfected vs. IL-37–transfected cells. n.s., not significant.

Fig. 3.

NLRP3 and ASC are required for the anti-inflammatory function of IL-37. Immortalized bone marrow-derived macrophages from WT mice (A) or ASC-deficient mice (ASC^−/−) (B) and NLRP3-deficient mice (NLPR3^−/−) (C) (9) were transfected with an IL-37 expression vector or an empty vector for control and then stimulated with LPS (100 ng/mL) or vehicle for 24 h. Mean ± SEM abundance of IL-6 and IL-1β (ratio of cytokines per milligram of protein of cell lysates) in the supernatants is shown (n = 5). *P < 0.05 and **P < 0.01 for empty vector vs. IL-37 expression plasmid.

Fig. 4.

Release of IL-37 from RAW macrophages, PBMCs, and DCs. (A) RAW–IL-37 cells were stimulated with LPS (100 ng/mL) or vehicle for 4 h and then subjected to a 20-min pulse of ATP (5 mM), as indicated, in the presence or absence of a pan-caspase inhibitor (pci, 10 μg/mL), followed by immunoblotting for IL-37. (B) PBMCs (5 × 10⁶ cells) were stimulated with LPS (100 ng/mL) or vehicle for 4 h, followed by treatment with ATP (5 mM) for 15 min. Supernatants of RAW–IL-37 cells and PBMCs were harvested, concentrated, and analyzed by Western blotting for the presence of IL-37. (C) Western blot analysis with murine anti-human IL-37 antibody of cytoplasmic extracts and concentrated supernatants of DCs stimulated with vehicle or anti-CD94, anti-HLA I, or anti-intercellular adhesion molecule 1 (ICAM-1) antibodies. One of three to four representative experiments performed is shown.

Fig. 5.

NK cell/DC interaction induces IL-37 clustering to the immunological synapse. Confocal microscopy analysis of tubulin (green) and IL-37 (red) in DC/NK cell cocultures after 6 h of incubation. (Upper and Lower) Two different focus layers of the same DC/NK cell conjugate are shown. Arrowheads and triangles indicate the immunological synapse where IL-37 and tubulin polarize together. One of four representative experiments performed is shown. (Magnification: 100×.)

Fig. 6.

Anti–IL-37 abrogates the protective effect of IL-37 in endotoxemia. (A) Human M1 macrophages were incubated with recombinant precursor IL-37 (1 ng/mL) premixed with 50 ng/mL mAb or the isotype control IgG for 1 h at room temperature and then added to the cells. After 2 h, LPS (10 ng/mL) was added, and supernatant IL-6 was determined after 24 h of incubation. Mean ± SEM percentage of change in IL-6 abundance is depicted. **P < 0.01 for IL-37 vs. IL-37 plus blocking antibody. (B) IL-37 transgenic mice were injected with the mAb against IL-37 or control IgG2B (100 μg) 3 h before LPS injection (100 μg per mouse, n = 5 mice per group). Mean ± SEM serum levels of IL-6 after 4 h of LPS treatment are shown. *P < 0.05 for IL-37 mAb vs. control IgG. (C) IL-37 transgenic and WT C57/Bl6 mice were injected with preimmune goat IgG (control) or polyclonal anti–IL-37 (200 μg) 3 h before LPS injection (100 μg per mouse, n = 3 mice per group). IL-6 was measured in sera after 4 h of LPS treatment. Data are expressed as fold increases of IL-6 (mean ± SEM) in mice treated with anti–IL-37 vs. control IgG. **P < 0.01 for C57/Bl6 vs. IL-37tg mice.

Fig. 7.

IL-37 mechanism of action as a dual-function cytokine.