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The extracellular matrix protein fibronectin (FN) is alternatively spliced in a variety of inflammatory conditions, resulting in increased inclusion of alternative exons EIIIA and EIIIB. Inclusion of these exons affects fibril formation, fibrosis, and inflammation. To define upstream regulators of alternative splicing in FN, we have developed an in vitro flow-cytometry based assay, using RNA-binding probes to determine alternative exon inclusion level in aortic endothelial cells. This approach allows us to detect exon inclusion in the primary transcripts themselves, rather than in surrogate splicing reporters. We validated this assay in cells with and without FN-EIIIA and -EIIIB expression. In a small-scale CRISPR KO screen of candidate regulatory splice factors, we successfully detected known regulators of EIIIA and EIIIB splicing, and detected several novel regulators. Finally, we show the potential in this approach to broadly interrogate upstream signaling pathways in aortic endothelial cells with a genome-wide CRISPR-KO screen, implicating the TNFalpha and RIG-I-like signaling pathways and genes involved in the regulation of fibrotic responses. Thus, we provide a novel means to screen the regulation of splicing of endogenous transcripts, and predict novel pathways in the regulation of FN-EIIIA inclusion. [ABSTRACT FROM AUTHOR]

The peripheral immune system plays a critical role in aging and in the response to brain injury. Emerging data suggest inflammatory responses are exacerbated in older animals following ischemic stroke; however, our understanding of these age-related changes is poor. In this work, we demonstrate marked differences in the composition of circulating and infiltrating leukocytes recruited to the ischemic brain of old male mice after stroke compared to young male mice. Blood neutrophilia and neutrophil invasion into the brain were increased in aged animals. Relative to infiltrating monocyte populations, brain-invading neutrophils had reduced phagocytic potential, and produced higher levels of reactive oxygen species and extracellular matrix-degrading enzymes (i.e., MMP-9), which were further exacerbated with age. Hemorrhagic transformation was more pronounced in aged versus young mice relative to infarct size. High numbers of myeloperoxidase-positive neutrophils were found in postmortem human brain samples of old (> 71 years) acute ischemic stroke subjects compared to non-ischemic controls. Many of these neutrophils were found in the brain parenchyma. A large proportion of these neutrophils expressed MMP-9 and positively correlated with hemorrhage and hyperemia. MMP-9 expression and hemorrhagic transformation after stroke increased with age. These changes in the myeloid response to stroke with age led us to hypothesize that the bone marrow response to stroke is altered with age, which could be important for the development of effective therapies targeting the immune response. We generated heterochronic bone marrow chimeras as a tool to determine the contribution of peripheral immune senescence to age- and stroke-induced inflammation. Old hosts that received young bone marrow (i.e., Young → Old) had attenuation of age-related reductions in bFGF and VEGF and showed improved locomotor activity and gait dynamics compared to isochronic (Old → Old) controls. Microglia in young heterochronic mice (Old → Young) developed a senescent-like phenotype. After stroke, aged animals reconstituted with young marrow had reduced behavioral deficits compared to isochronic controls, and had significantly fewer brain-infiltrating neutrophils. Increased rates of hemorrhagic transformation were seen in young mice reconstituted with aged bone marrow. This work suggests that age alters the immunological response to stroke, and that this can be reversed by manipulation of the peripheral immune cells in the bone marrow. [ABSTRACT FROM AUTHOR]

Naïve antigen-specific CD8 T cells expand in response to infection and can be phenotypically separated into distinct effector populations, which include memory precursor effector cells (MPECs) and short-lived effector cells (SLECs). In the days before the peak of the T cell response, a third population called early effector cells (EECs) predominate the antigen-specific response. However, the contribution of the EEC population to the CD8 T cell differentiation program during an antimicrobial immune response is not well understood. To test if EEC populations were pre-committed to either an MPEC or SLEC fate, we purified EECs from mice infected with Listeria monocytogenes (LM) or vesicular stomatitis virus (VSV), where the relative frequency of each population is known to be different at the peak of the response. Sorted EECs transferred into uninfected hosts revealed that EECs were pre-programmed to differentiate based on early signals received from the distinct infectious environments. Surprisingly, when these same EECs were transferred early into mismatched infected hosts, the transferred EECs could be diverted from their original fate. These results delineate a model of differentiation where EECs are programmed to form MPECs or SLECs, but remain susceptible to additional inflammatory stimuli that can alter their fate. [ABSTRACT FROM AUTHOR]

Atherosclerosis is the major cause of ischemic heart disease and stroke, the leading causes of mortality worldwide. The central pathological features of atherosclerosis include macrophage infiltration and foam cell formation. However, the detailed mechanisms regulating these two processes remain unclear. Here we show that oxidative stress-activated Ca 2+ -permeable transient receptor potential melastatin 2 (TRPM2) plays a critical role in atherogenesis. Both global and macrophage-specific Trpm2 deletion protect Apoe -/- mice against atherosclerosis. Trpm2 deficiency reduces oxidized low-density lipoprotein (oxLDL) uptake by macrophages, thereby minimizing macrophage infiltration, foam cell formation and inflammatory responses. Activation of the oxLDL receptor CD36 induces TRPM2 activity, and vice versa. In cultured macrophages, TRPM2 is activated by CD36 ligands oxLDL and thrombospondin-1 (TSP1), and deleting Trpm2 or inhibiting TRPM2 activity suppresses the activation of CD36 signaling cascade induced by oxLDL and TSP1. Our findings establish the TRPM2-CD36 axis as a molecular mechanism underlying atherogenesis, and suggest TRPM2 as a potential therapeutic target for atherosclerosis., Competing Interests: Competing Interests All authors declare no competing interests.