The role of HMGB1 in the pathogenesis of inflammatory and autoimmune diseases.

High-mobility group box 1 (HMGB1) protein is a highly abundant protein that can promote the pathogenesis of inflammatory and autoimmune diseases once it is in an extracellular location. This translocation can occur with immune cell activation as well as cell death, with the conditions for release associated with the expression of different isoforms. These isoforms result from post-translational modifications, with the redox states of three cysteines at positions 23, 45 and 106 critical for activity. Depending on the redox states of these residues, HMGB1 can induce cytokine production via toll-like receptor 4 (TLR4) or promote chemotaxis by binding the chemokine CXCL12 for stimulation via CXCR4. Fully oxidized HMGB1 is inactive. During the course of inflammatory disease, HMGB1 can therefore play a dynamic role depending on its redox state. As a mechanism to generate alarmins, cell death is an important source of HMGB1, although each major cell death form (necrosis, apoptosis, pyroptosis and NETosis) can lead to different isoforms of HMGB1 and variable levels of association of HMGB1 with nucleosomes. The association of HMGB1 with nucleosomes may contribute to the pathogenesis of systemic lupus erythematosus by producing nuclear material whose immunological properties are enhanced by the presence of an alarmin. Since HMGB1 levels in blood or tissue are elevated in many inflammatory and autoimmune diseases, this molecule can serve as a unique biomarker as well as represent a target of novel therapies to block its various activities.

Neonatal FcR overexpression boosts humoral immune response in transgenic mice.

The neonatal FcR (FcRn) regulates IgG and albumin homeostasis, mediates maternal IgG transport, takes active part in phagocytosis, and delivers Ag for presentation. We have previously shown that overexpression of FcRn in transgenic (Tg) mice extends the half-life of mouse IgG by reducing its clearance. In this paper, we demonstrate that immunization of these mice with OVA and trinitrophenyl-conjugated human IgG results in a 3- to 10-fold increase of Ag-specific IgM and IgG in serum. The IgM increase was unexpected because FcRn does not bind IgM. Our results showed that the affinity of the Ag-specific IgG was at least as good in Tg mice as in the wild-type (wt) controls, implying appropriate affinity maturation in both groups. Influenza vaccination produced a 2-fold increase in the amount of virus-specific Ab in Tg animals, which proved twice as efficient in a hemagglutination inhibition assay as was the case in wt controls. After immunization, Tg mice displayed significantly larger spleens containing a higher number of Ag-specific B cells and plasma cells, as well as many more granulocytes and dendritic cells, analyzed by ELISPOT and flow cytometric studies. The neutrophils from these Tg mice expressed the Tg FcRn and phagocytosed IgG immune complexes more efficiently than did those from wt mice. These results show that FcRn overexpression not only extends the IgG half-life but also enhances the expansion of Ag-specific B cells and plasma cells. Although both effects increase the level of Ag-specific IgG, the increase in immune response and IgG production seems to be more prominent compared with the reduced IgG clearance.

RAGE inhibitor TTP488 (Azeliragon) suppresses metastasis in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a highly aggressive and metastatic cancer subtype, which is generally untreatable once it metastasizes. We hypothesized that interfering with the Receptor for Advanced Glycation End-products (RAGE) signaling with the small molecule RAGE inhibitors (TTP488/Azeliragon and FPS-ZM1) would impair TNBC metastasis and impair fundamental mechanisms underlying tumor progression and metastasis. Both TTP488 and FPS-ZM1 impaired spontaneous and experimental metastasis of TNBC models, with TTP488 reducing metastasis to a greater degree than FPS-ZM1. Transcriptomic analysis of primary xenograft tumor and metastatic tissue revealed high concordance in gene and protein changes with both drugs, with TTP488 showing greater potency against metastatic driver pathways. Phenotypic validation of transcriptomic analysis by functional cell assays revealed that RAGE inhibition impaired TNBC cell adhesion to multiple extracellular matrix proteins (including collagens, laminins, and fibronectin), migration, and invasion. Neither RAGE inhibitor impaired cellular viability, proliferation, or cell cycle in vitro. Proteomic analysis of serum from tumor-bearing mice revealed RAGE inhibition affected metastatic driver mechanisms, including multiple cytokines and growth factors. Further mechanistic studies by phospho-proteomic analysis of tumors revealed RAGE inhibition led to decreased signaling through critical BC metastatic driver mechanisms, including Pyk2, STAT3, and Akt. These results show that TTP488 impairs metastasis of TNBC and further clarifies the signaling and cellular mechanisms through which RAGE mediates metastasis. Importantly, as TTP488 displays a favorable safety profile in human studies, our study provides the rationale for evaluating TTP488 in clinical trials to treat or prevent metastatic TNBC.

The Alarmin Properties of DNA and DNA-associated Nuclear Proteins.

PURPOSE: The communication of cell injury and death is a critical element in host defense. Although immune cells can serve this function by elaborating cytokines and chemokines, somatic cells can repurpose nuclear macromolecules to function as damage-associated molecular patterns (DAMPs) or alarmins to exert similar activity. Among these molecules, DNA, high-mobility group box-1, and histone proteins can all act as DAMPs once they are in an extracellular location. This review describes current information on the role of the nuclear DAMPs, their translocation to the outside of cells, and pathways of activation after uptake into the inside of immune cells. METHODS: MEDLINE and PubMed databases were searched for citations (1990-2016) in English related to the following terms: DAMPs, high-mobility group box-1, DNA, histones, cell death, danger, and immune activation. Selected articles with the most relevant studies were included for a more detailed consideration. FINDINGS: Although nuclear molecules have important structural and genetic regulatory roles inside the cell nucleus, when released into the extracellular space during cell death, these molecules can acquire immune activity and serve as alarmins or DAMPs. Although apoptosis is generally considered the source of extracellular nuclear material, other cell death pathways such as necroptosis, NETosis, and pyroptosis can contribute to the release of nuclear molecules. Importantly, the release of nuclear DAMPs occurs with both soluble and particulate forms of these molecules. The activity of nuclear molecules may depend on posttranslational modifications, redox changes, and the binding of other molecules. Once in an extracellular location, nuclear DAMPs can engage the same pattern recognition receptors as do pathogen-associated molecular patterns. These interactions can activate immune cells and lead to cytokine and chemokine production. Among these receptors, internal receptors for DNA are key to the response to this molecule; the likely function of these internal sensors is the recognition of DNA from intracellular infection by bacteria or viruses. Activation of these receptors requires translocation of extracellular DNA into specialized compartments. In addition to nuclear DNA, mitochondrial DNA can also serve as a DAMP. IMPLICATIONS: The communication of cell injury and death is a critical element in host defense and involves the repurposing of nuclear molecules as immune triggers. As such, the presence of extracellular nuclear material can serve as novel biomarkers for conditions involving cell injury and death. Targeting of these molecules may also represent an important new approach to therapy.

Double-stranded RNA binding may be a general plant RNA viral strategy to suppress RNA silencing.

In plants, RNA silencing (RNA interference) is an efficient antiviral system, and therefore successful virus infection requires suppression of silencing. Although many viral silencing suppressors have been identified, the molecular basis of silencing suppression is poorly understood. It is proposed that various suppressors inhibit RNA silencing by targeting different steps. However, as double-stranded RNAs (dsRNAs) play key roles in silencing, it was speculated that dsRNA binding might be a general silencing suppression strategy. Indeed, it was shown that the related aureusvirus P14 and tombusvirus P19 suppressors are dsRNA-binding proteins. Interestingly, P14 is a size-independent dsRNA-binding protein, while P19 binds only 21-nucleotide ds-sRNAs (small dsRNAs having 2-nucleotide 3' overhangs), the specificity determinant of the silencing system. Much evidence supports the idea that P19 inhibits silencing by sequestering silencing-generated viral ds-sRNAs. In this study we wanted to test the hypothesis that dsRNA binding is a general silencing suppression strategy. Here we show that many plant viral silencing suppressors bind dsRNAs. Beet yellows virus Peanut P21, clump virus P15, Barley stripe mosaic virus gammaB, and Tobacco etch virus HC-Pro, like P19, bind ds-sRNAs size-selectively, while Turnip crinkle virus CP is a size-independent dsRNA-binding protein, which binds long dsRNAs as well as ds-sRNAs. We propose that size-selective ds-sRNA-binding suppressors inhibit silencing by sequestering viral ds-sRNAs, whereas size-independent dsRNA-binding suppressors inactivate silencing by sequestering long dsRNA precursors of viral sRNAs and/or by binding ds-sRNAs. The findings that many unrelated silencing suppressors bind dsRNA suggest that dsRNA binding is a general silencing suppression strategy which has evolved independently many times.