Dissertation > Excellent graduate degree dissertation topics show

Crucial Role of HMGB1 in Activation of Macrophage and Pathogenesis of SLE Induced by Activated Lymphocyte Derived-DNA

Author: YuShanShan
Tutor: XiongSiDong
School: Fudan University
Course: Immunology
Keywords: SLE ALD-DNA HMGB1 Macrophage
CLC: R392
Type: Master's thesis
Year: 2011
Downloads: 35
Quote: 0
Read: Download Dissertation

Abstract

Systemic lupus erythematosus (SLE) is a severe multisystemic autoimmune disease manifested by inflammatory damage in a variety of organs. It is characterized by the generation of pathogenic antibodies responded to a variety of autoantigens including nuclear and cytoplasmic antigens such as double-stranded DNA (dsDNA), nucleosomes, and complement activation. However, native mammalian DNA is generally considered to be poorly immunogenic or even non-immunogenic. Therefore, searching for the autoantigens which were responsible for the development of SLE has long been the subject of investigation.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 BALB/c mice with genomic DNA derived from ConA-stimulated syngeneic splenocytes evoked SLE-like syndrome, including high levels of anti-dsDNA antibody production, proteinuria and glomerulonephritis. Further study showed that ALD-DNA could activate macrophages to produce large amounts of inflammatory cytokines, which were implicated in SLE pathogenesis. However, the mechanism underlying the activation of macrophages induced by ALD-DNA remains to be elucidated.HMGB1 is a nuclear protein that modulates chromatin accessibility and is present at variable levels in most cells. HMGB1 is also known as amphoterin because of the presence of two basic DNA-binding domains called box A and B, and an acidic C-terminal tail. It has been reported that HMGB1 serves as a universal sentinel for nucleic acids that is required for the full-blown, nucleic-acid-induced activation of innate immune responses mediated by the more discriminative pattern recognition receptors. Recently, accumulating data indicated that HMGB1 might play an important role in autoimmune rheumatic diseases. However, the potential role of HMGB1 in the pathogenesis of SLE induced by ALD-DNA remains unclear. In the present study, we evaluated the potential role of HMGB1 in the activation of macrophages and the onset of SLE induced by ALD-DNA, which might further our understanding of SLE pathogenesis and provide clues for developing novel clinical therapies against SLE.Part I HMGB1 was crucial for ALD-DNA induced activation of macrophagesWe first confirmed that ALD-DNA could efficiently activate macrophages. We showed that ALD-DNA could upregulate the expression of co-stimulatory molecules including CD80, CD86 and MHC classⅡon both bone marrow derived macrophages (BMDMs) and macrophage cell line RAW264.7 cells. Furthermore, ALD-DNA could induce macrophages to produce abundant IL-6, IL-12 and TNF-α. In contrast, UnALD-DNA failed to induce the activation of macrophages. Meanwhile, we found that HMGB1 was secreted from macrophages when stimulated with ALD-DNA. The secretion of HMGB1 was induced by ALD-DNA in a dose-dependent manner. The level of extracellular HMGB1 in the supernatant was positively correlated with the production of inflammatory cytokines. These data suggested that extracellular HMGB1 might play a role in the activation of macrophages induced by ALD-DNA.To elucidate the potential role of HMGB1 in the activation of macrophages induced by ALD-DNA, the macrophages were transfected with siRNA against HMGB1 and the production of inflammatory cytokines were assayed. We found that down-regulation of HMGB1 could dramatically abrogate the production of inflammatory cytokines induced by ALD-DNA. Importantly, we found that the A box peptide, an antagonist of extracellular HMGB1, could inhibit the induction of inflammatory cytokines in a dose-dependent manner. We did not observe any activation of macrophages while treated with pure HMGB1 protein alone or plus the UnALD-DNA. However, we found that HMGB1 could act in synergy with ALD-DNA to activate macrophages. These results indicated that extracellular HMGB1 was critical for ALD-DNA to activate macrophages.We used both competitive ELISA and circular dichroism spectra to indicate that HMGB1 could directly bind to ALD-DNA to form an immune complex. To further explore the underlying mechanism responsible for the production of inflammatory cytokines induced by the HMGB1-ALD-DNA complex, the potential role of TLR2, TLR4 and RAGE which have been proposed as HMGB1 receptors were determined. We found that the TLR2/4 inhibitor had no significant effect on the induction of inflammatory cytokines. In contrast, both the siRNA against RAGE and the RAGE-Fc could significantly abrogate the induction of inflammatory cytokines, suggesting that RAGE was responsible for recognizing HMGB1 to mediate the activation of macrophages induced by ALD-DNA.In concIusion, our data demonstrated that the extracellular HMGB1 conferred ALD-DNA to induce the macrophage activation. Notably, we found that the recognition of RAGE to HMGB1 was responsible for HMGB1-ALD-DNA complex to activate macrophages.PartⅡHMGB1 was critical for ALD-DNA induced onset of SLEGiven the closely correlation between abnormal macrophage activation and the pathogenesis of SLE, we sought to investigate the potential role of HMGB1 in the pathogenesis of SLE induced by ALD-DNA. We found that extracellular HMGB1 was significantly elevated in the serum of ALD-DNA immunized mice. To further elucidate the potential role of HMGB1 in the pathogenesis of SLE induced by ALD-DNA, groups of BALB/c mice were administrated with HMGB1 expression plasmids or extracellular HMGB1 antagonist glycyrrhizin, and then immunized with ALD-DNA. We found that, overexpression of HMGB1 resulted in elevated level of serological anti-dsDNA antibodies and urine protein, as well as more severed renal histopathology in ALD-DNA immunized mice. In contrast, the glycyrrhizin treated group showed impaired induction of anti-dsDNA antibodies, accompanied by slightly proteinuria and renal histopathology. These findings suggested that HMGB1 was essential for ALD-DNA to induce SLE autoimmune disease in SLE-non-susceptible mice. Finally, we found that ALD-DNA induced significantly higher levels of proinflammatory cytokines from macrophages isolated from HMGB1 overespression immunized mice than that from glycyrrhizin treated immunized mice, indicating that HMGB1 conferred the response of macrophages to ALD-DNA, which might partly explain their distinct severity of SLE-like syndrome. Altogether, our study found that HMGB1, acting as an innate DNA sensor, is up-regulated in macrophages by ALD-DNA and could bind with ALD-DNA and trigger macrophage activation via RAGE pathway. Our data would facilitate our further understanding of the SLE pathogenesis and be helpful for developing novel strategies for SLE therapies.

Crucial Role of HMGB1 in Activation of Macrophage and Pathogenesis of SLE Induced by Activated Lymphocyte Derived-DNA

Abstract

Related Dissertations