Author Correction: The kinase TBK1 controls IgA class switching by negatively regulating noncanonical NF-κB signaling.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The kinase TBK1 controls IgA class switching by negatively regulating noncanonical NF-κB signaling.

Immunoglobulin class switching is crucial for the generation of antibody diversity in humoral immunity and, when deregulated, also has severe pathological consequences. How the magnitude of immunoglobulin isotype switching is controlled is still poorly understood. Here we identify the kinase TBK1 as a pivotal negative regulator of class switching to the immunoglobulin A (IgA) isotype. B cell-specific ablation of TBK1 in mice resulted in uncontrolled production of IgA and the development of nephropathy-like disease signs. TBK1 negatively regulated IgA class switching by attenuating noncanonical signaling via the transcription factor NF-κB, an action that involved TBK1-mediated phosphorylation and subsequent degradation of the NF-κB-inducing kinase NIK. Our findings establish TBK1 as a pivotal negative regulator of the noncanonical NF-κB pathway and identify a unique mechanism that controls IgA production.

The ubiquitin ligase Peli1 negatively regulates T cell activation and prevents autoimmunity.

T cell activation is subject to tight regulation to avoid inappropriate responses to self antigens. Here we show that genetic deficiency in the ubiquitin ligase Peli1 caused hyperactivation of T cells and rendered T cells refractory to suppression by regulatory T cells and transforming growth factor-ß (TGF-ß). As a result, Peli1-deficient mice spontaneously developed autoimmunity characterized by multiorgan inflammation and autoantibody production. Peli1 deficiency resulted in the nuclear accumulation of c-Rel, a member of the NF-κB family of transcription factors with pivotal roles in T cell activation. Peli1 negatively regulated c-Rel by mediating its Lys48 (K48) ubiquitination. Our results identify Peli1 as a critical factor in the maintenance of peripheral T cell tolerance and demonstrate a previously unknown mechanism of c-Rel regulation.

Noncanonical NF-κB pathway controls the production of type I interferons in antiviral innate immunity.

Production of type I interferons (IFN-I) is a crucial innate immune mechanism against viral infections. IFN-I induction is subject to negative regulation by both viral and cellular factors, but the underlying mechanism remains unclear. We report that the noncanonical NF-κB pathway was stimulated along with innate immune cell differentiation and viral infections and had a vital role in negatively regulating IFN-I induction. Genetic deficiencies in major components of the noncanonical NF-κB pathway caused IFN-I hyperinduction and rendered cells and mice substantially more resistant to viral infection. Noncanonical NF-κB suppressed signal-induced histone modifications at the Ifnb promoter, an action that involved attenuated recruitment of the transcription factor RelA and a histone demethylase, JMJD2A. These findings reveal an unexpected function of the noncanonical NF-κB pathway and highlight an important mechanism regulating antiviral innate immunity.

OTUD7B controls non-canonical NF-κB activation through deubiquitination of TRAF3.

The non-canonical NF-κB pathway forms a major arm of NF-κB signalling that mediates important biological functions, including lymphoid organogenesis, B-lymphocyte function, and cell growth and survival. Activation of the non-canonical NF-κB pathway involves degradation of an inhibitory protein, TNF receptor-associated factor 3 (TRAF3), but how this signalling event is controlled is still unknown. Here we have identified the deubiquitinase OTUD7B as a pivotal regulator of the non-canonical NF-κB pathway. OTUD7B deficiency in mice has no appreciable effect on canonical NF-κB activation but causes hyperactivation of non-canonical NF-κB. In response to non-canonical NF-κB stimuli, OTUD7B binds and deubiquitinates TRAF3, thereby inhibiting TRAF3 proteolysis and preventing aberrant non-canonical NF-κB activation. Consequently, the OTUD7B deficiency results in B-cell hyper-responsiveness to antigens, lymphoid follicular hyperplasia in the intestinal mucosa, and elevated host-defence ability against an intestinal bacterial pathogen, Citrobacter rodentium. These findings establish OTUD7B as a crucial regulator of signal-induced non-canonical NF-κB activation and indicate a mechanism of immune regulation that involves OTUD7B-mediated deubiquitination and stabilization of TRAF3.

A rapid method for quantifying cytoplasmic versus nuclear localization in endogenous peripheral blood leukocytes by conventional flow cytometry.

A biochemical system and method have been developed to enable the quantitative measurement of cytoplasmic versus nuclear localization within cells in whole blood. Compared with the analyses of nuclear localization by western blot or fluorescence microscopy, this system saves a lot of time and resources by eliminating the necessity of purification and culturing steps, and generates data that are free from the errors and artifacts associated with using tumor cell lines or calculating nuclear signals from 2D images. This user-friendly system enables the analysis of cell signaling within peripheral blood cells in their endogenous environment, including measuring the kinetics of nuclear translocation for transcription factors without requiring protein modifications. We first demonstrated the efficiency and specificity of this system for targeting nuclear epitopes, and verified the results by fluorescence microscopy. Next, the power of the technique to analyze LPS-induced signaling in peripheral blood monocytes was demonstrated. Finally, both FoxP3 localization and IL-2-induced STAT5 signaling in regulatory T cells were analyzed. We conclude that this system can be a useful tool for enabling multidimensional molecular-biological analyses of cell signaling within endogenous peripheral blood cells by conventional flow cytometry. © 2017 International Society for Advancement of Cytometry.

Automated leukocyte processing by microfluidic deterministic lateral displacement.

We previously developed a Deterministic Lateral Displacement (DLD) microfluidic method in silicon to separate cells of various sizes from blood (Davis et al., Proc Natl Acad Sci 2006;103:14779-14784; Huang et al., Science 2004;304:987-990). Here, we present the reduction-to-practice of this technology with a commercially produced, high precision plastic microfluidic chip-based device designed for automated preparation of human leukocytes (white blood cells; WBCs) for flow cytometry, without centrifugation or manual handling of samples. After a human blood sample was incubated with fluorochrome-conjugated monoclonal antibodies (mAbs), the mixture was input to a DLD microfluidic chip (microchip) where it was driven through a micropost array designed to deflect WBCs via DLD on the basis of cell size from the Input flow stream into a buffer stream, thus separating WBCs and any larger cells from smaller cells and particles and washing them simultaneously. We developed a microfluidic cell processing protocol that recovered 88% (average) of input WBCs and removed 99.985% (average) of Input erythrocytes (red blood cells) and >99% of unbound mAb in 18 min (average). Flow cytometric evaluation of the microchip Product, with no further processing, lysis or centrifugation, revealed excellent forward and side light scattering and fluorescence characteristics of immunolabeled WBCs. These results indicate that cost-effective plastic DLD microchips can speed and automate leukocyte processing for high quality flow cytometry analysis, and suggest their utility for multiple other research and clinical applications involving enrichment or depletion of common or rare cell types from blood or tissue samples. © 2016 International Society for Advancement of Cytometry.

A Novel Semiconductor-Based Flow Cytometer with Enhanced Light-Scatter Sensitivity for the Analysis of Biological Nanoparticles.

The CytoFLEX is a novel semiconductor-based flow cytometer that utilizes avalanche photodiodes, wavelength-division multiplexing, enhanced optics, and diode lasers to maximize light capture and minimize optical and electronic noise. Due to an increasing interest in the use of extracellular vesicles (EVs) as disease biomarkers, and the growing desire to use flow cytometry for the analyses of biological nanoparticles, we assessed the light-scatter sensitivity of the CytoFLEX for small-particle detection. We found that the CytoFLEX can fully resolve 70 nm polystyrene and 98.6 nm silica beads by violet side scatter (VSSC). We further analyzed the detection limit for biological nanoparticles, including viruses and EVs, and show that the CytoFLEX can detect viruses down to 81 nm and EVs at least as small as 65 nm. Moreover, we could immunophenotype EV surface antigens, including directly in blood and plasma, demonstrating the double labeling of platelet EVs with CD61 and CD9, as well as triple labeling with CD81 for an EV subpopulation in one donor. In order to assess the refractive indices (RIs) of the viruses and EVs, we devised a new method to inversely calculate the RIs using the intensity vs. size data together with Mie-theory scatter efficiencies scaled to reference-particle measurements. Each of the viruses tested had an equivalent RI, approximately 1.47 at 405 nm, which suggests that flow cytometry can be more broadly used to easily determine virus sizes. We also found that the RIs of EVs increase as the particle diameters decrease below 150 nm, increasing from 1.37 for 200 nm EVs up to 1.61 for 65 nm EVs, expanding the lower range of EVs that can be detected by light scatter. Overall, we demonstrate that the CytoFLEX has an unprecedented level of sensitivity compared to conventional flow cytometers. Accordingly, the CytoFLEX can be of great benefit to virology and EV research, and will help to expand the use of flow cytometry for minimally invasive liquid biopsies by allowing for the direct analysis of antigen expression on biological nanoparticles within patient samples, including blood, plasma, urine and bronchoalveolar lavages.

Tumor Necrosis Factor Receptor Associated Factors (TRAFs) 2 and 3 Form a Transcriptional Complex with Phosho-RNA Polymerase II and p65 in CD40 Ligand Activated Neuro2a Cells.

The tumor necrosis factor receptor-associated factors (TRAFs) have been classically described as adaptor proteins that function as solely cytosolic signaling intermediates for the TNF receptor superfamily, Toll-like receptors (TLRs), NOD, like receptors (NLRs), cytokine receptors, and others. In this study, we show for the first time that TRAFs are present within the cytoplasm and nucleus of Neuro2a cells and primary cortical neurons, and that TRAF2 and TRAF3 translocate into the nucleus within minutes of CD40L stimulation. Analysis of the transcriptional regulatory potential of TRAFs by luciferase assay revealed that each of the TRAFs differentially functions as a transcriptional activator or repressor in a cell-specific manner. Interestingly, ChIP-qPCR data demonstrate that TRAFs 2/3, p65, and pRNAPol II form part of a transcriptional complex on the Icam-1 gene promoter upon CD40L stimulation. We further determined that TRAF2 recruitment to the nucleus is critical for the ubiquitination of H2b, a transcription permissive epigenetic modification. Our findings demonstrate for the first time that TRAFs 2/3 participate in the formation of a CD40L-induced transcriptional complex in neuronal cells.

TPL2 mediates autoimmune inflammation through activation of the TAK1 axis of IL-17 signaling.

Development of autoimmune diseases, such as multiple sclerosis and experimental autoimmune encephalomyelitis (EAE), involves the inflammatory action of Th1 and Th17 cells, but the underlying signaling mechanism is incompletely understood. We show that the kinase TPL2 is a crucial mediator of EAE and is required for the pathological action of Th17 cells. TPL2 serves as a master kinase mediating the activation of multiple downstream pathways stimulated by the Th17 signature cytokine IL-17. TPL2 acts by linking the IL-17 receptor signal to the activation of TAK1, which involves a dynamic mechanism of TPL2-TAK1 interaction and TPL2-mediated phosphorylation and catalytic activation of TAK1. These results suggest that TPL2 mediates TAK1 axis of IL-17 signaling, thereby promoting autoimmune neuroinflammation.

Peli1 promotes microglia-mediated CNS inflammation by regulating Traf3 degradation.

Microglia are crucial for the pathogenesis of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). Here we show that the E3 ubiquitin ligase Peli1 is abundantly expressed in microglia and promotes microglial activation during the course of EAE induction. Peli1 mediates the induction of chemokines and proinflammatory cytokines in microglia and thereby promotes recruitment of T cells into the central nervous system. The severity of EAE is reduced in Peli1-deficient mice despite their competent induction of inflammatory T cells in the peripheral lymphoid organs. Notably, Peli1 regulates Toll-like receptor (TLR) pathway signaling by promoting degradation of TNF receptor-associated factor 3 (Traf3), a potent inhibitor of mitogen-activated protein kinase (MAPK) activation and gene induction. Ablation of Traf3 restores microglial activation and CNS inflammation after the induction of EAE in Peli1-deficient mice. These findings establish Peli1 as a microglia-specific mediator of autoimmune neuroinflammation and suggest a previously unknown signaling mechanism of Peli1 function.