MRP1 Transporter Regulates Interleukin-1β Secretion By Glutathionylation

Background and aimsCytokine storm (cytokine storm) is the phenomenon of a variety of cytokines being produced rapidly, which could lead to multiple organ failure. These cytokines include interleukin-1β (IL-1β), IL-6 and interferon gamma(IFN-y). IL-1β is a pro-inflammatory cytokine, which plays an important role in infection, injury and tumor development. In many diseases IL-1β levels is significantly increased, such as familial Mediterranean fever, familial cold autoinflammatory syndrome, Muckle-Wells syndrome, neonatal onset multisystem inflammatory disease, hyperimmunoglobulin D syndrome, and adult-onset Still disease, as well as in myeloid leukemia. In view of the important clinical value of IL-1β, it is necessary to make a deep study on the secretion mechanism of IL-1β.Currently, little is known about IL-1β secretion. IL-1β is produced in its inactive form proIL-1β (31kDa), then cleaved by caspase-1 into the active matureIL-1β(17kDa). Because IL-1β lacks of signal peptide, it can not be secreted into the extracellular environment through the classical endoplasmic reticulum-Golgi apparatus secretory pathway. What the mechanisms are actually involved in the IL-1β secretion process remains poorly defined. As IL-1β is evenly distributed in the cytoplasm, we speculatethat the extensive presence of various transporters on cytomembrane could be the main secretory pathway of IL-1β. In this study, we will screen the unknown transporters related to IL-1β secretion, and explore its molecular mechanism.Methods1 IL-1β secretion from human and murine monocytes, macrophages and neutrophils1.1 Acquisition of human and murine monocytes, macrophages and neutrophilsWe collected and cultured human monocytes, macrophages and polymorphonuclear cells; obtained the PMA-induced macrophages derived from the differentiated THP-1 cells; collected mouse peritoneal and bone marrow macrophages; cultured mouse macrophage cell line RAW264.7 cell and J774A.1 cell; collected mouse peritoneal and bone marrow neutrophils; cultured and differentiated HL60 cells into neutrophils. Cells purity were determined using Wright-Giemsa staining and flow cytometry.1.2 IL-1β secretion testWe used lμg/ml LPS 4h+ATP1mM 45min to stimulate cells and set up three groups:Untreated cell group(UT), LPS stimulation group(L), LPS+ATP double stimulation group(LA). For IL-1β secretion test, we used ELISA, Western Blotting and immnunofluorescence assay.2 Screen transporters related to IL-1β secretion2.1 Using inhibitors to screen transporters related to IL-1β secretionWe purchased inhibitors specific for MRP1, BCRP, P-gp, OATP1B1and MATE transporters, to screen which kind of transporters were related to IL-1β secretion.2.2 MRP1 function examination using SNARF1 and MRP1 protein expression testLet 10μg/ml SNARF1 loaded for 30 minutes, then unloaded for 7h, at the end of unloading we used FACS to analyze the intracellular SNARF1 percentage. For MRP1 expression, we used FACS, Western Blotting and immunofluorescence assay.2.3 IL-1β secretion in MRP1 knock out miceWe purchased FVB wild type mice(control) and MRP1 knock out mice and cultured their bone marrow macrophage to do IL-1β level testing.3 Screen transporters related to IL-1β secretion3.1 Peritonitis modelWe introperitoneally injected (i.p.) different LPS concentrations into mice, with negative control, lmg/kg,1.5mg/kg,2mg/kg,5mg/kg, 10mg/kg, and 50mg/kg to create mouse peritonitis model.3.2 32 multiplex assay of MRPlko miceWe i.p. LPS lmg/kg into MRPlko mice, then performed 32 multiplex assay, included Eotaxin, G-CSF, GM-CSF, IFN-y, IL-la, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17, IP-10, KC, LIF, LIX, MCP-1, M-CSF, MIG,MP-1α, MIP-1β, MIP-2, RANTES, TNF-a and VEGF.3.3 Survival rates of miceMice were i.p. with 15 mg/kg LPS, then these mice were blocked with anakinra 745mg/kg or/and IL-18 BPd lmg/kg to check the survival rate, with unblocked mice as negative controls.4 Glutathionylation checking of IL-1β in living cellsAfter BioGEE labeled all glutathionylated intracellular proteins, we lysed cell to get cell lysate, used goat-anti-human IL-1β antibody to pull-down IL-1β protein from the cell lysates by immunoprecipitation. For Western Blotting, we used rabbit-anti-human IL-1β as the first antibody to check the efficiency of pull-down, and used streptavidin-HRP as the first antibody to determine the glutathionylation level of intracellular IL-1 p.Results1 IL-1β secretion on human and murine monocytes, macrophages and neutrophils1.1 Cultured human and murine monocytes, macrophages and neutrophilsWe obtained the human monocytes, macrophages and polymorphonuclear cells. The PMA induced macrophages differentiated from THP-1 cells were obtained. Mouse peritoneal and bone marrow macrophages were obtained either by injecting and flushing mouse peritoneal cavity with appropriate amount of medium or bone marrow aspiration. The mouse macrophage cell lines RAW264.7 and J774A.1 cells were purchased from company and cultured in media according to the instructions of company. The mouse peritoneal and bone marrow neutrophils were separated from either peritoneal flushing media or bone marrow aspiration. The human neutrophils were differentiated from HL60 cells and cultured with RPMI 1640 medium. The cell types were comfirmed by observation under the microscope and flow ctometry after Wright-Giemsa staining and immunofluorescent staining.1.2 IL-1β secretion patternMonocyes and neutrophils only need LPS single stimulation to release matureIL-1β, while macrophages need LPS and ATP double stimulation to release matureIL-1β. THP-1 derived macrophage, J774A.1 cell and RAW264.7cell could secret proIL-1β.2 MRP1 transporters related to IL-1β secretion2.1 Inhibit MRP1 transporter could decrease IL-1β secretionAmong MRP1, BCRP, P-gp, OATP1B1 and MATE transporters, only inhibiting MRP1 transporter could significantly reduce IL-1β secretion levels, indicating that the MRP1 transporters were closely related to IL-1β secretion..2.2 MRP1 transporter worked well and expressed on cytoplasmic membraneHigh intracellular SNARF1 percentage was only found in MRP1 inhibited group. By using FACS, Western Blotting and immunofluorescence assay, we successfully detected MRP1 protein.2.3 MRP1 knock OutUsing mice bone marrow MO macrophages, the secreted mature IL-lp level in MRP1ko mice were lower than those of FVBwt mice (P=0.044).3 Blocking IL-1β level could increase survival rate of mice3.1 Peritonitis model workedPeritoneal injection of LPS lmg/kg could cause peritonitis in mice, and mice injected with this dose could survive for more than one month. After checking, LPS 5mg/kg and 10mg/kg showed to be sublethal doses, while 50mg/kg was lethal dose. During the period of peritonitis, many types of inflammatory cytokines were increased in mice, including IL-1β.3.2 Blocked IL-1β level increased survival rate of miceMice were ip. LPS with a dose of 15 mg/kg. Compared to unblocked group, anakinra 745mg/kg single blocking group, anakinra 745mg/kg+IL-18 BPd lmg/kg double blocking group both showed better survival rate.4 Intracellular IL-1P was glutathionylatedThe Western Blotting result of rabbit-anti-human IL-1β showed that IL-1β was pulled down, and the Western Blotting result of streptavidin-HRP proved that the intracellular IL-1βp was glutathionylated.Conclusion(1) Monocyes and neutrophils only needed single stimulation (LPS) to release matureIL-1βp, while macrophages needed double stimulation (LPS+ATP) to release matureIL-1β. THP-1 derived human macrophage, mouse macrophage cell lines J774A.1 cells and RAW264.7cells could secret proIL-1β.(2) MRP1 transporter expressed on cytoplasmic membrane, and was one pathway of IL-1β secretion.(3) During inflammation, the secretion of IL-1β was increased. Blocking IL-1β could improve the survival rate of inflamed mice.(4) Intracellular IL-1β was glutathionylated, and MRP1 transporter may regulate IL-1β secretion by glutathionylation.