Weiwen Jiang

The relationship between apoptosis and high-mobility group protein 1 release from murine macrophages stimulated with lipopolysaccharide or polyinosinic-polycytidylic acid

High-mobility group protein 1 (HMGB1) is a nonhistone nuclear protein whose function depends on cellular location. Inside the cell, HMGB1 modulates a variety of important cellular processes, including transcription, whereas outside the cell, HMGB1 acts as a cytokine that can promote inflammation and mediate sepsis and arthritis in animal models. In in vitro studies, proinflammatory molecules such as LPS, lipoteichoic acid, polyinosinic-polycytidylic acid (poly(I:C)), TNF-α, and type I and II IFNs can induce HMGB1 release from macrophages. Although these agents can activate cells, they can also induce apoptosis under certain circumstances. Therefore, because of evidence that apoptotic as well as necrotic cells can contribute to HMGB1-mediated events in sepsis, we have investigated the relationship between apoptosis and HMGB1 release in macrophages and other cells. In these experiments, using RAW 264.7 cells as a model, LPS and poly(I:C) caused HMGB1 release into the medium whereas CpG ODN failed to induce this response. With both LPS and poly(I:C), the extent of HMGB1 release correlated with the occurrence of apoptosis as measured by caspase 3 activation, lactate dehydrogenase release, and TUNEL staining. Similar results were obtained with primary murine macrophages as well as human Jurkat T cells. For Jurkat cells, poly(I:C) and NO donors induced apoptosis as well as HMGB1 release. Together, these results indicate that HMGB1 release from macrophages is correlated with the occurrence of apoptosis and suggest that these processes reflect common mechanisms and can occur concomitantly.

The Role of IFN-α and Nitric Oxide in the Release of HMGB1 by RAW 264.7 Cells Stimulated with Polyinosinic-Polycytidylic Acid or Lipopolysaccharide

High mobility group protein 1 (HMGB1) is a nonhistone nuclear protein with a dual function. Inside the cell, HMGB1 binds to DNA and modulates a variety of processes, including transcription. Outside the cell, HMGB1 displays cytokine activity and can promote inflammation, serving as a mediator in models of shock and arthritis. In in vitro studies, proinflammatory molecules such as LPS, lipoteichoic acid, dsRNA, TNF-α, and IFN-γ can induce HMGB1 release from macrophages. To define further the release process, we investigated the role of the downstream mediators, NO and IFN-α, in the release of HMGB1 from RAW 264.7 macrophage cells stimulated with LPS or polyinosinic-polycytidylic acid (poly(I:C)). In these experiments, 1400W, an inhibitor of NO production by the inducible NO synthase, reduced HMGB1 release stimulated by LPS, but not poly(I:C), whereas neutralizing IFN-α prevented HMGB1 release induced by poly(I:C), but not LPS. The addition of an NO donor and rIFN-α to RAW 264.7 cells caused HMGB1 release. Furthermore, inhibition of JNK activation attenuated HMGB1 release induced by either LPS or poly(I:C). Analysis of bone marrow-derived macrophages stimulated by LPS or poly(I:C) showed patterns of HMGB1 release similar to those of RAW 264.7 cells. Together, these experiments indicate that, although both LPS and poly(I:C) induce HMGB1 release from RAW 264.7 cells and murine macrophages, the response is differentially dependent on NO and IFN-α.

Expression of high mobility group protein 1 in the sera of patients and mice with systemic lupus erythematosus

High mobility group protein 1 (HMGB1) is a non-histone nuclear protein with a dual function. Inside the cell, HMGB1 binds to DNA and modulates a variety of processes, including transcription. Outside the cell, HMGB1 can serve as an alarmin to mediate disease manifestations in animal models of sepsis and arthritis; in these models, blocking HMGB1 can attenuate disease.1–3 In in vitro experiments, HMGB1 translocation and cellular release can occur during activation as well as cell death and is present in tissue in conditions, such as rheumatoid arthritis and cutaneous lupus.4 5 While original studies suggested that release occurs only with necrosis, …

The effects of CpG DNA on HMGB1 release by murine macrophage cell lines

DNA containing cytosine‐guanine dinucleotide (CpG) motifs (CpG DNA) has potent immunostimulatory activities that resemble those of lipopolysaccharide (LPS) in its effects on the innate immune system. Among its activities, LPS can induce the release of high mobility group protein (HMGB1) by macrophages, a dual function molecule that can mediate the late effects of LPS. To determine whether CpG DNA can also induce HMGB1 release, the effects of a synthetic CpG oligonucleotide (ODN) on HMGB1 release from RAW 264.7 and J774A.1 cells were assessed by Western blotting of culture supernatants. Under conditions in which the CpG ODN activated the cell lines, as assessed by stimulation of tumor necrosis factor α and interleukin‐12, it failed to cause HMGB1 release into the media. Although unable to induce HMGB1 release by itself, the CpG ODN nevertheless potentiated the action of LPS. With RAW 264.7 cells, lipoteichoic acid and polyinosinic‐polycytidylic acid, like LPS, stimulated HMGB1 release as well as cytokine production. These results indicate that the effects of CpG DNA on macrophages differ from other ligands of Toll‐like receptors and may lead to a distinct pattern of immune cell activation in the context of infection or its use as an immunomodulatory agent.

Pivotal Advance: Inhibition of HMGB1 nuclear translocation as a mechanism for the anti-rheumatic effects of gold sodium thiomalate

Gold compounds such as gold sodium thiomalate (GST) can reduce the symptoms of rheumatoid arthritis (RA), although their mechanism of action is not well defined. As the proinflammatory mediator high mobility group box chromosomal protein 1 (HMGB1) may play a role in the pathogenesis of RA, we have performed in vitro studies to investigate whether GST inhibits HMGB1 release as the basis of its mode of action. Murine RAW 264.7 or human THP‐1 macrophage cells were stimulated in culture with agents causing extracellular HMGB1 release, including LPS, IFN‐γ, polyinosinic:polycytidylic acid, IFN‐β, or NO in the presence of GST, ranging from 0 μM to 250 μM. Secretion and intracellular location of HMGB1 were assessed by Western blotting, HMGB1‐specific ELISPOT assay, and immunofluorescent staining. In parallel, TNF and IFN‐β levels were analyzed by ELISPOT and/or ELISA. Supernatant NO production was analyzed by the Griess method. At pharmacologically relevant doses, GST inhibited the extracellular release of HMGB1 from activated macrophages and caused the nuclear retention of this protein; in contrast, no effects were observed on the secretion or production of TNF. Release of the key endogenous mediators of HMGB1 translocation, IFN‐β and NO, was inhibited by GST. This inhibition required gold, as sodium thiomalate did not affect the responses measured. Furthermore, gold chloride also inhibited release of HMGB1. Together, these results suggest a new mechanism for the anti‐rheumatic effects of gold salts in RA and the potential of drugs, which interfere with intracellular HMGB1 transport mechanisms, as novel agents to treat RA.

Mechanisms of Disease: the role of high-mobility group protein 1 in the pathogenesis of inflammatory arthritis.

High-mobility group protein 1 (HMG1) is a nonhistone nuclear protein that is a prototype of a dual-function alarmin whose immune activity is dependent upon its cellular location. Inside the cell, HMG1 binds to DNA and has a role in transcriptional regulation. Outside the cell, HMG1 acts as a cytokine and has activities that resemble those of tumor necrosis factor. The cytokine activities of HMG1 become manifest when this protein translocates from the nucleus to the cytoplasm and, eventually, into the external milieu; this translocation occurs during cell activation and cell death. Given its cytokine activity, HMG1 has been implicated in the pathogenesis of a broad range of immune-mediated diseases including arthritis. The role for this protein in arthritis was established by observations of the expression of HMG1 in synovial tissue of patients with rheumatoid arthritis as well as in the joints of animals used to model arthritis. Furthermore, in the mouse model of collagen-induced arthritis, treatment with antibodies to HMG1 or to an inhibitory domain of HMG1 can attenuate joint inflammation and damage. These studies identify a novel pathway in the pathogenesis of inflammatory arthritis, as well as a new target for biologic therapy.

A Toll-Like Receptor 7, 8, and 9 Antagonist Inhibits Th1 and Th17 Responses and Inflammasome Activation in a Model of IL-23-Induced Psoriasis

Psoriasis is a chronic inflammatory skin disease that involves the induction of T-helper 1 (Th1) and T-helper 17 (Th17) cell responses and the aberrant expression of proinflammatory cytokines, including IL-1β. Copious evidence suggests that abnormal activation of Toll-like receptors (TLRs) contributes to the initiation and maintenance of psoriasis. We have evaluated an antagonist of TLR7, 8, and 9 as a therapeutic agent in an IL-23-induced psoriasis model in mice. Psoriasis-like skin lesions were induced in C57BL/6 mice by intradermal injection of IL-23 in the ear or dorsum. IL-23-induced increase in ear thickness was inhibited in a dose-dependent manner by treatment with antagonist. Histological examination of ear and dorsal skin tissues demonstrated a reduction in epidermal hyperplasia in mice treated with the antagonist. Treatment with antagonist also reduced the induction of Th1 and Th17 cytokines in skin and/or serum, as well as dermal expression of inflammasome components, NLRP3 and AIM2, and antimicrobial peptides. These results indicate that targeting TLR7, 8, and 9 may provide a way to neutralize multiple inflammatory pathways that are involved in the development of psoriasis. The antagonist has the potential for the treatment of psoriasis and other autoimmune diseases.

Role of toll-like receptors in the pathogenesis of dystrophin-deficient skeletal and heart muscle

Although the cause of Duchenne muscular dystrophy (DMD) is known, the specific factors that initiate and perpetuate disease progression are not well understood. We hypothesized that leaky dystrophin-deficient skeletal muscle releases endogenous danger signals (TLR ligands), which bind to Toll-like receptors (TLRs) on muscle and immune cells and activate downstream processes that facilitate degeneration and regeneration in dystrophic skeletal muscle. Here, we demonstrate that dystrophin-deficient mouse muscle cells show increased expression of several cell-surface and endosomal TLRs. In vitro screening identified ssRNA as a relevant endogenous TLR7 ligand. TLR7 activation led to myd88-dependent production of pro-inflammatory cytokines in dystrophin-deficient muscle cells, and cause significant degeneration/regeneration in vivo in mdx mouse muscle. Also, knockout of the central TLR adaptor protein, myd88 in mdx mice significantly improved skeletal and cardiac muscle function. Likewise, proof-of-concept experiments showed that treating young mdx mice with a TLR7/9 antagonist significantly reduced skeletal muscle inflammation and increased muscle force, suggesting that blocking this pathway may have therapeutic potential for DMD.

Role of Toll‐like Receptors in HMGB1 Release from Macrophages

Abstract:  HMGB1 is a nonhistone nuclear protein that can serve as a cytokine and activate innate immunity. The translocation of this molecule from the inside to the outside of cells is a critical event in inflammation, occurring following activation of certain Toll‐like receptors (TLRs) as well as during the course of apoptotic as well as necrotic cell death. Because the kinetics of HMGB1 release differs from that of a conventional cytokine, it provides a broader therapeutic window and may be an important new target of therapy for inflammatory, autoimmune, and infectious diseases.

Identification of Novel Innate Immune Genes by Transcriptional Profiling of Macrophages Stimulated with TLR Ligands

Toll-like receptors (TLRs) are key receptors in innate immunity and trigger responses following interaction with pathogen-associated molecular patterns (PAMPs). TLR3, TLR4 and TLR9 recognize double stranded RNA, lipopolysaccharide (LPS) and CpG DNA, respectively. These receptors differ importantly in downstream adaptor molecules. TLR4 signals through MyD88 and TRIF; in contrast, the TLR3 pathway involves only TRIF while TLR9 signals solely through MyD88. To determine how differences in downstream signaling could influence gene expression in innate immunity, gene expression patterns were determined for the RAW264.7 macrophage cell line stimulated with LPS, poly (I:C), or CpG DNA. Gene expression profiles 6 and 24h post-stimulation were analyzed to determine genes, pathways and transcriptional networks induced. As these experiments showed, the number and extent of genes expressed varied with stimulus. LPS and poly (I:C) induced an abundant array of genes in RAW264.7 cells at 6h and 24h following treatment while CpG DNA induced many fewer. By analyzing data for networks and pathways, we prioritized differentially expressed genes with respect to those common to the three TLR ligands as well as those shared by LPS and poly (I:C) but not CpG DNA. The importance of changes in gene expression was demonstrated by experiments indicating that RNA interference-mediated inhibition of two genes identified in this analysis, PLEC1 and TPST1, reduced IL-6 production by J774A.1 and RAW264.7 macrophages stimulated with LPS. Together, these findings delineate macrophage gene response patterns induced by different PAMPs and identify new genes that have not previously been implicated in innate immunity.