Increased concentrations of DNA-containing immune complexes in the serum are associated with systemic autoimmune diseases such as lupus. Stimulation of Toll-like receptor 9 (TLR9) by DNA is important in the activation of plasmacytoid dendritic cells and B cells. Here we show that HMGB1, a nuclear DNA-binding protein released from necrotic cells, was an essential component of DNA-containing immune complexes that stimulated cytokine production through a TLR9-MyD88 pathway involving the multivalent receptor RAGE. Moreover, binding of HMGB1 to class A CpG oligodeoxynucleotides considerably augmented cytokine production by means of TLR9 and RAGE. Our data demonstrate a mechanism by which HMGB1 and RAGE activate plasmacytoid dendritic cells and B cells in response to DNA and contribute to autoimmune pathogenesis.

Autoantibodies directed against citrullinated epitopes of proteins are highly diagnostic of rheumatoid arthritis (RA), and elevated levels of protein citrullination can be found in the joints of patients with RA. Calcium-dependent peptidyl-arginine deiminases (PAD) are the enzymes responsible for citrullination. PAD2 and PAD4 are enriched in neutrophils and likely drive citrullination under inflammatory conditions. PADs may be released during NETosis or cell death, but the mechanisms responsible for PAD activity under physiological conditions have not been fully elucidated. To understand how PADs citrullinate extracellular proteins, we investigated the cellular localization and activity of PAD2 and PAD4, and we report that viable neutrophils from healthy donors have active PAD4 exposed on their surface and spontaneously secrete PAD2. Neutrophil activation by some stimulatory agents increased the levels of immunoreactive PAD4 on the cell surface, and some stimuli reduced PAD2 secretion. Our data indicate that live neutrophils have the inherent capacity to express active extracellular PADs. These novel pathways are distinguished from intracellular PAD activation during NETosis and calcium influx-mediated hypercitrullination. Our study implies that extracellular PADs may have a physiological role under non-pathogenic conditions as well as a pathological role in RA.

Our previous studies have shown that isolated cytotoxic T lymphocyte (CTL), B-cell, and T-helper epitopes, for which we coined the term minigenes, can be effective vaccines; when expressed from recombinant vaccinia viruses, these short immunogenic sequences confer protection against a variety of viruses and bacteria. In addition, we have previously demonstrated the utility of DNA immunization using plasmids encoding full-length viral proteins. Here we combine the two approaches and evaluate the effectiveness of minigenes in DNA immunization. We find that DNA immunization with isolated minigenes primes virus-specific memory CTL responses which, 4 days following virus challenge, appear similar in magnitude to those induced by vaccines known to be protective. Surprisingly, this vigorous CTL response fails to confer protection against a normally lethal virus challenge, although the CTL appear fully functional because, along with their high lytic activity, they are similar in affinity and cytokine secretion to CTL induced by virus infection. However this DNA immunization with isolated minigenes results in a low CTL precursor frequency; only 1 in ∼40,000 T cells is epitope specific. In contrast, a plasmid encoding the same minigene sequences covalently attached to the cellular protein ubiquitin induces protective immunity and a sixfold-higher frequency of CTL precursors. Thus, we show that the most commonly employed criterion to evaluate CTL responses—the presence of lytic activity following secondary stimulation—does not invariably correlate with protection; instead, the better correlate of protection is the CTL precursor frequency. Recent observations indicate that certain effector functions are active in memory CTL and do not require prolonged stimulation. We suggest that these early effector functions of CTL, immediately following infection, are critical in controlling virus dissemination and in determining the outcome of the infection. Finally, we show that improved performance of the ubiquitinated minigenes most probably requires polyubiquitination of the fusion protein, suggesting that the enhancement results from more effective delivery of the minigene to the proteasome.

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Background Diabetic nephropathy (DN) is a severe end-stage kidney disease developed from diabetes mellitus. The involvement of circular RNA (circRNAs) in the regulation of DN pathogenesis has been implied, but the underlying mechanism of DN is still lacking. This study aimed to investigate the effect of hsa_circ_0003928 on the inflammation and apoptosis of high glucose (HG)-induced renal tubular cells. Methods The expression of hsa_circ_0003928, miR-151-3p and Anxa2 in blood samples from DN patients and healthy controls was detected by RT-qPCR. Human renal epithelial cells HK-2 were incubated with D-glucose (30 mmol/l) to establish DN model in vitro. RT-qPCR analysis confirmed the transfection effects and detected the expressions of TNF-α, IL-6 and IL-1β. Western blotting analysis determined the protein expression of Anxa2, Bcl-2, Bax, cleaved caspase-3 and caspase-3. The production of ROS was detected by DCF-DA method and production of inflammatory cytokines was verified by ELISA assay. CCK-8 assay and TUNEL assay were performed to determine cell viability and apoptosis, respectively. Dual-luciferase reporter assay was performed to confirm the relationship between miR-151-3p and hsa_circ_0003928 or Anxa2. Results Hsa_circ_0003928 and Anxa2 mRNA levels were increased, whereas miR-151-3p was decreased in both HG-induced HK-2 cells and patients with DN. Hsa_circ_0003928 knockdown could decrease cell viability loss and apoptosis, increase Bcl-2 expression, and decrease Bax and cleaved caspase-3 expression. Besides, hsa_circ_0003928 knockdown suppressed HG-induced overproduction of ROS, TNF-α, IL-6 and IL-1β. However, the effects made by miR-151-3p inhibition were opposite to those made by hsa_circ_0003928 knockdown. Furthermore, the binding sites between miR-151-3p and hsa_circ_0003928 or Anxa2 were predicted and verified. Protein expression of Anxa2 was suppressed by hsa_circ_0003928 knockdown, which was rescued by miR-151-3p inhibition. Conclusion These results demonstrated that hsa_circ_0003928 could act as a sponge of miR-151-3p and regulate HG-induced inflammation and apoptosis partly through regulating Anxa2.