Elevated Levels of Interferon-α Act Directly on B Cells to Breach Multiple Tolerance Mechanisms Promoting Autoantibody Production.

OBJECTIVE: Elevated levels of serum interferon-α (IFNα) and the disruption of B cell tolerance are central to systemic lupus erythematosus (SLE) immunopathogenesis; however, the relationship between these 2 processes remains unclear. The purpose of this study was to investigate the impact of elevated IFNα levels on B cell tolerance mechanisms in vivo and determine whether any changes observed were due to the direct effect of IFNα on B cells. METHODS: Two classical mouse models of B cell tolerance were used in conjunction with an adenoviral vector encoding IFNα to mimic the sustained elevations of IFNα seen in SLE. The role of B cell IFNα signaling, T cells, and Myd88 signaling was determined using B cell-specific IFNα receptor-knockout, CD4+ T cell-depleted, or Myd88-knockout mice, respectively. Flow cytometry, enzyme-linked immunosorbent assay, real-time quantitative polymerase chain reaction, and cell cultures were used to study the effects of elevated IFNα on the immunologic phenotype. RESULTS: Elevation of serum IFNα disrupts multiple B cell tolerance mechanisms and leads to autoantibody production. This disruption was dependent upon B cell expression of IFNα receptor. Many of the IFNα-mediated alterations also required the presence of CD4+ T cells as well as Myd88, suggesting that IFNα acts directly on B cells to modify their response to Myd88 signaling and their ability to interact with T cells. CONCLUSION: The results provide evidence that elevated IFNα levels act directly on B cells to facilitate autoantibody production and further highlight the importance of IFN signaling as a potential therapeutic target in SLE.

Treatment with Angiotensin-(1-7) Prevents Development of Oral Papilloma Induced in K-ras Transgenic Mice.

Oral Squamous Cell Carcinoma (OSCC) is the most common malignant cancer affecting the oral cavity. It is characterized by high morbidity and very few therapeutic options. Angiotensin (Ang)-(1-7) is a biologically active heptapeptide, generated predominantly from AngII (Ang-(1-8)) by the enzymatic activity of angiotensin-converting enzyme 2 (ACE 2). Previous studies have shown that Ang-(1-7) counterbalances AngII pro-tumorigenic actions in different pathophysiological settings, exhibiting antiproliferative and anti-angiogenic properties in cancer cells. However, the prevailing effects of Ang-(1-7) in the oral epithelium have not been established in vivo. Here, we used an inducible oral-specific mouse model, where the expression of a tamoxifen-inducible Cre recombinase (CreERtam), which is under the control of the cytokeratin 14 promoter (K14-CreERtam), induces the expression of the K-ras oncogenic variant KrasG12D (LSLK-rasG12D). These mice develop highly proliferative squamous papilloma in the oral cavity and hyperplasia exclusively in oral mucosa within one month after tamoxifen treatment. Ang-(1-7) treated mice showed a reduced papilloma development accompanied by a significant reduction in cell proliferation and a decrease in pS6 positivity, the most downstream target of the PI3K/Akt/mTOR signaling route in oral papilloma. These results suggest that Ang-(1-7) may be a novel therapeutic target for OSCC.

Genetic engineering in primary human B cells with CRISPR-Cas9 ribonucleoproteins.

Genome editing in human cells with targeted nucleases now enables diverse experimental and therapeutic genome engineering applications, but extension to primary human B cells remains limited. Here we report a method for targeted genetic engineering in primary human B cells, utilizing electroporation of CRISPR-Cas9 ribonucleoproteins (RNPs) to introduce gene knockout mutations at protein-coding loci with high efficiencies that in some cases exceeded 80%. Further, we demonstrate knock-in editing of targeted nucleotides with efficiency exceeding 10% through co-delivery of oligonucleotide templates for homology directed repair. We delivered Cas9 RNPs in two distinct in vitro culture systems to achieve editing in both undifferentiated B cells and activated B cells undergoing differentiation, reflecting utility in diverse experimental conditions. In summary, we demonstrate a powerful and scalable research tool for functional genetic studies of human B cell biology that may have further applications in engineered B cell therapeutics.

Oxidized Low-Density Lipoprotein Loading of Macrophages Downregulates TLR-Induced Proinflammatory Responses in a Gene-Specific and Temporal Manner through Transcriptional Control.

Hypercholesterolemia is a key risk factor for atherosclerosis and leads to the uptake of native and oxidized low-density lipoprotein (oxLDL) by macrophages (Mϕs) and foam cell formation. Inflammatory processes accompany Mϕ foam cell formation in the artery wall, yet the relationship between Mϕ lipid loading and their response to inflammatory stimuli remains elusive. We investigated proinflammatory gene expression in thioglycollate-elicited peritoneal Mϕs, bone marrow-derived Mϕs and dendritic cells, and RAW264.7 cells. Loading with oxLDL did not induce peritoneal Mϕ apoptosis or modulate basal-level expression of proinflammatory genes. Upon stimulation of TLR4, the rapid induction of IFN-ß was inhibited in cells loaded with oxLDL, whereas the induction of other proinflammatory genes by TLR4 (LPS), TLR3 (polyriboinosinic-polyribocytidylic acid), TLR2 (Pam3CSK4), and TLR9 (CpG) remained comparable within the first 2 h. Subsequently, the expression of a subset of proinflammatory genes (e.g., IL-1ß, IL-6, CCL5) was reduced in oxLDL-loaded cells at the level of transcription. This phenomenon was partially dependent on NF erythroid 2-related factor 2 (NRF2) but not on nuclear liver X receptors α and ß (LXRα,ß), peroxisome proliferator-activated receptor-γ (PPARγ), and activating transcription factor 3 (ATF3). LPS-induced NF-κB reporter activity and intracellular signaling by NF-κB and MAPK pathways were comparable in oxLDL-loaded Mϕs, yet the binding of p65/RelA (the prototypic NF-κB family member) was reduced at IL-6 and CCL5 promoters. This study revealed that oxLDL loading of Mϕs negatively regulates transcription at late stages of TLR-induced proinflammatory gene expression and implicates epigenetic mechanisms such as histone deacetylase activity.