The Role And The Mechanism Of HMGB1in The Macrophage Activation Induced By Activated Lymphocyte Derived-DNA

Objective:Systemic lupus erythematosus (SLE) is a kind of typical autoimmune diseases characterized by the production of a variety of antinuclear antibodies, especially anti-double-stranded (ds) DNA antibodies. Currently, the pathogenesis of SLE is not yet entirely clear, associated with many factors. Under normal circumstances, native mammalian DNA is generally considered to be poorly immunogenic and even non-immunogenic. Therefore, exploring the underlying mechanism of how to break the immune tolerance to self DNA to activate the immune system is much more important in study of the pathogenesis of SLE.During the effect to study the pathogenesis of SLE, our previous study has established a lupus model in BALB/c mice induced by syngeneic activated lymphocyte-derived DNA (ALD-DNA) but not un-activated lymphocyte-derived DNA (UnALD-DNA). Immunization of naive mice with genomic DNA derived from ConA-stimulated syngeneic splenocytes evoked the emblematical autoimmune syndrome such as high level of anti-dsDNA Abs, glomerulonephritis and proteinuria, while genomic DNA from un-activated splenocytes failed to induce this syndrome. Further research revealed that ALD-DNA induced the production of inflammatory cytokines in immune cells particularly macrophages. It has been reported that macrophages are important cells involved in innate immunity, but also a class of specific APC, playing an important role in both innate immunity and adaptive immunity. However, how ALD-DNA activates the macrophages and what the specific mechanism remain undefined.High mobility group protein B1(HMGB1) is a member of the high mobility group (HMG) proteins, with a high migration rate in polyacrylamide gel electrophoresis (SDS-PAGE), ubiquitously expressed in eukaryotic cells.In its usual location, as a nuclear molecule, HMGB1has been implicated in diverse intracellular functions, including the stabilization of nucleosomal structure and the facilitation of gene transcription. HMGB1acts as an important damage-associated molecular pattern after its release, which can occur actively by secretion from activated immune cells such as macrophages and monocytes, or passively from dead cells. It has been reported that HMGB1could combined with a variety of nucleic acid components, recognized by nucleic acid specific receptors to activate the downstream signaling pathway, which play a significant role in the immune process of response to nucleic acid components. In recent years, studies have found that HMGB1is considered to be a new kind of inflammatory cytokines as it activates inflammatory cells to induce secretion of cytokines, and is causing widespread concern in domestic and foreign scholars.Previous studies in our laboratory found that HMGB1was involved in ALD-DNA-induced macrophage activation, but the mechanism of HMGB1mediated in the process remains unclear. Either cytoplasmic or secreted into the extracellular HMGB1plays an i mportant role in theidentification, uptake and intracellular transport of ALD-DNA, whic h are worthy to be studied. In this study, we explored the underlying mechanism of HMGB1involved in the ALD-DNA induced macrophage activation, focusing on the location of HMGB1mediated the macrohage activation induced by ALD-DNA. This research will not only help us to better underatand the mechanism of HMGB1promoted the macrophage activation induced by ALD-DNA and to elucidate the pathogenesis of SLE, providing a more solid theoretical basis for the prevention and in favor of searching for a new target for SLE.Methods:To analyzed the expression of extracellular HMGB1in RAW264.7cells in the response to ALD-DNA, we first stimulated macrophage cell line RAW264.7with50μg/ml ALD-DNA or UnALD-DNA for24h, then the level of HMGB1in supernatants were assayed by ELISA and the expression of HMGB1in cell lysate was detected by western blot. To elucidate the potential role of HMGB1in ALD-DNA induced macrophage activation, we generated HMGB1stable knockdown cell lines from the murine macrophage cell line RAW264.7, with target-specific small hairpin RNA (shRNA) sequences against HMGB1and confirmed the knockdown efficiency by qRT-PCR and western blot. Next, we stimulated RAW264.7and HMGB1KD RAW264.7with50μg/ml ALD-DNA or UnALD-DNA for24h, the expression of co-stimulatory molecules and MHC class II molecule were assayed by flow cytometry and IL-6, IL-10, TNF-a mRNA detected by real time.Furthermore, to explore the role of extracellular HMGB1in ALD-DNA induced macrophage activation, ethyl pyruvate was used to inhibit the release of HMGB1from macrophages. The extracellular HMGB1in supernatants in response to ALD-DNA stimulation was determined by ELISA, while the intracellular localization of HMGB1was observed by laser scanning confocal microscope. In addition, A box peptide and anti-HMGB1neutralizing antibody were used to block the function of extracellular HMGB1,following treatment, IL-6, IL-10, TNF-a mRNA were detected by real time and ELIS A. At the same time, we added HMGB1protein into the medium and assayed the levels of inflammatory cytokines.Finally, to study the underlying mechanism of HMGB1mediated in ALD-DNA-induced macrophage activation, we first determined the interaction of HMGB1and ALD-DNA or UnALD-DNA by competitive ELISA;second, the effect of HMGB1on the uptake of ALD-DNA or UnALD-DNA were determined by flow cytometry; last, laser scanning confocal microscope was used to observe the effects of HMGB1on vesicle transport of ALD-DNA or UnALD-DNA.Results:We found that the expression of extracellular HMGB1was significantly increased in the supernatants of RAW264.7cells treated with ALD-DNA than that in control groups and it is time-and dose-dependent. In contrast, the expression of HMGB1in the lysate of RAW264.7cells was generally comparable between the ALD-DNA stimulation group and the control groups. Next, we generated HMGB1stable knockdown cell lines from the murine macrophage cell line RAW264.7, with target-specific small hairpin RNA (shRNA) sequences against HMGB1. The release of HMGB1was inhibited by knocking down HMGB1. Besides, ALD-DNA-induced macrophage cell surface costimulatory molecules, MHC class Ⅱ molecule showed decreased expression, mRNA expression of inflammatory cytokines was also significantly reduced. In addition, Ethyl pyruvate was used to prevent the secretion of HMGB1, we revealed that the extracellular HMGB1in supernatants in response to ALD-DNA stimulation was significantly downregulated and HMGB1accumulation in the cytoplasm occurred. Consistently, Ethyl pyruvate significantly decreased the expression of inflammatory cytokines in the cell supernatants. To further confirm these results, we also block the function of HMGB1by administration of A box peptide and anti-HMGB1neutralizing antibody, finding that incubation of A box peptide and anti-HMGB1neutralizing antibody could abrogated the ALD-DNA induced macrohage activation in a dose dependent manner. Given that extracellular HMGB1was crucial for ALD-DNA induced macrophage activation, we next analyzed whether HMGB1protein alone was capable to exert the similar function. Compared with ALD-DNA, we found that HMGB1protein alone had a weaker but significant positive effect on the production of inflammatory cytokines in a dose dependent manner. Furthermore, we found that HMGB1protein could elevate the effect of ALD-DNA to induce the activation of macrophages.We further explored the mechanism of HMGB1mediated the macrophage activation induced by ALD-DNA. First, we found that HMGB1bind to ALD-DNA or UnALD-DNA with different binding affinity by indirect competitive ELISA. The affinity of HMGB1binding to ALD-DNA is significantly higher than that to UnALD-DNA. Second, we found that the effect of HMGB1on the macrophage activation induced by ALD-DNA is not related to the uptake of ALD-DNA or UnALD-DNA by macrophages. Third, HMGB1can significantly increase the localization of ALD-DNA at the endosome, and accelerates the recruitment of ALD-DNA to the endosomal vesicles.Conclusion:ALD-DNA can effectively stimulate macrophages to secrete HMGB1in a time and dose dependent manner. Extracellular HMGB1plays a pivotal role in the macrophage activation induced by ALD-DNA. Once secreted into the extracellular matrix, HMGB1can bind to ALD-DNA to form a complex, which in turn increased and acceleratedto the transport to endosomal vesicles, thereby enhancing the ALD-DNA-induced macrophage activation to promote pathological development of SLE. This study was aimed to elucidate the potential role of HMGB1in the activation of macrophages induced by ALD-DNA and the underlying mechanism, to look forward to providing a more solid theoretical basis for the pathogenesis of SLE.