Simultaneous polychromatic flow cytometric detection of multiple forms of regulated cell death.

Currently the study of Regulated Cell Death (RCD) processes is limited to the use of lysed cell populations for Western blot analysis of each separate RCD process. We have previously shown that intracellular antigen flow cytometric analysis of RIP3, Caspase-3 and cell viability dye allowed the determination of levels of apoptosis (Caspase-3+ ve/RIP3- ve), necroptosis (RIP3Hi + ve/Caspase-3- ve) and RIP1-dependent apoptosis (Caspase-3+ ve/RIP3+ ve) in a single Jurkat cell population. The addition of more intracellular markers allows the determination of the incidence of parthanatos (PARP), DNA Damage Response (DDR, H2AX), H2AX hyper-activation of PARP (H2AX/PARP) autophagy (LC3B) and ER stress (PERK), thus allowing the identification of 124 sub-populations both within live and dead cell populations. Shikonin simultaneously induced Jurkat cell apoptosis and necroptosis the degree of which can be shown flow cytometrically together with the effects of blockade of these forms of cell death by zVAD and necrostatin-1 have on specific RCD populations including necroptosis, early and late apoptosis and RIP1-dependent apoptosis phenotypes in live and dead cells. Necrostatin-1 and zVAD was shown to modulate levels of shikonin induced DDR, hyper-action of PARP and parthanatos in the four forms of RCD processes analysed. LC3B was up-regulated by combined treatment of zVAD with chloroquine which also revealed that DNA damage was reduced in live cells but enhanced in dead cells indicating the role of autophagy in maintaining cell health. This approach to RCD research should be a great advance to understanding the mechanisms of drugs and their effects upon RCD populations.

Simultaneous flow cytometric immunophenotyping of necroptosis, apoptosis and RIP1-dependent apoptosis.

Flow cytometry was been widely used to measure apoptosis for many decades but the researcher has no definitive way of determining other forms of cell death using this technology. The use of Western Blot technology has numerous drawbacks in that all the cells in the sample whether live, dead or maybe undergoing multiple discrete forms of cell death are analysed as one population. Flow cytometry given that it can analyse different sub-populations of cells within a sample would reveal the expression of cell death markers within these sub-populations rather than just give a single result from the entire population. Here we describe a flow cytometric assay fully realising that potential by the use of anti-RIP-3 (Receptor-interacting serine/threonine-protein kinase 3) and anti-active caspase-3 fluorescently tagged antibodies and a fixable live dead fluorescent dye. This allows the determination of the degree of necroptosis, apoptosis and RIP1-dependent apoptosis within live and dead populations. Necroptosis was identified by the up-regulation of RIP3, while RIP1-dependent apoptosis was described by double positive for RIP3/active Caspase-3 events in live and dead populations. Apoptotic cells were defined by an active-Caspase-3+ve/RIP3-ve phenotype. Pan-caspase blocker zVAD and RIP1 inhibitors GSK'481 or necrostatin-1 revealed interesting modulations of such sub-populations of Jurkat cells. This novel flow cytometric assay employing two antibodies and a fixable viability probe provides the researcher with in-depth analysis of various forms of regulated forms of cell death beyond what is currently available and is a major methodological advancement in this field.

Association of innate immune activation with latent Epstein-Barr virus in active MS lesions.

OBJECTIVE: To determine whether the activation of innate immune responses, which can be elicited by pathogenic and endogenous triggers, is associated with the presence of Epstein-Barr virus (EBV) infection in the multiple sclerosis (MS) brain. METHODS: White matter postmortem MS (n = 10) and control tissue (n = 11) was analyzed for the expression of the proinflammatory cytokine interferon α (IFNα) by immunohistochemistry and for EBV by using the highly sensitive method of EBV-encoded RNA (EBER) in situ hybridization. RESULTS: We detected overexpression of IFNα in active areas of white matter MS lesions but not in inactive MS lesions, normal-appearing white matter, or normal brains. The presence of IFNα in macrophages and microglia (expressing human leukocyte antigen class II) is suggestive of local production as part of an acute inflammatory process. Interestingly, EBERs were also specifically detected in areas where IFNα was overexpressed in these preselected active MS lesions. EBER+ cells were also found in CNS lymphoma and stroke cases, but were absent in other control brains. We next addressed a potential mechanism, e.g., the role of EBERs in eliciting IFNα production, and transfected EBERs into human embryonic kidney (HEK) cells. We used HEK cells that stably expressed Toll-like receptor-3, which recognizes double-stranded RNAs, associated with many viral infections. EBERs elicited IFNα production in vitro. CONCLUSION: These findings suggest that latent EBV infection may contribute to the inflammatory milieu in active MS lesions by activating innate immune responses, e.g., IFNα production. Unraveling the underlying mechanisms may help in uncovering causal pathways and developing better treatment strategies for MS and other neuroinflammatory diseases.

Translational Mini-Review Series on B cell subsets in disease. Transitional B cells in systemic lupus erythematosus and Sjögren's syndrome: clinical implications and effects of B cell-targeted therapies.

Systemic lupus erythematosus (SLE) and Sjögren's syndrome are autoimmune disorders which are characterized by a disturbed B cell homeostasis which leads ultimately to dysfunction of various organs. One of the B cell subsets that appear in abnormal numbers is the population of transitional B cells, which is increased in the blood of patients with SLE and Sjögren's syndrome. Transitional B cells are newly formed B cells. In mice, transitional B cells undergo selection checks for unwanted specificity in the bone marrow and the spleen in order to eliminate autoreactive B cells from the circulating naive B cell population. In humans, the exact anatomical compartments and mechanisms of the specificity check-points for transitional B cells remain unclear, but appear to be defective in SLE and Sjögren's syndrome. This review aims to highlight the current understanding of transitional B cells and their defects in the two disorders before and after B cell-targeted therapies.

Antigen presenting cells and T cell interactions in the gastrointestinal tract.

Dendritic cells make up less than 1% of the cells in lymph nodes and tissues, but they are critical in initiating and directing T cell immune responses. In the gut, with exposure to myriad food and bacterial antigens, they probably control T cell unresponsiveness to food antigens and T cell hypersensitivity in disease situations. The need for the immune system to 'know' gut luminal antigens is demonstrated by the fact that dendritic cells send processes through the epithelium and directly sample antigens in the gut lumen. We present evidence that type I INF made by plasmacytoid dendritic cells may be important in coeliac disease.