Autoantibodies to components of apoptotic cells, such as anti-perlecan antibodies, contribute to rejection in organ transplant recipients. However, mechanisms of immunization to apoptotic components remain largely uncharacterized. We used large-scale proteomics, with validation by electron microscopy and biochemical methods, to compare the protein profiles of apoptotic bodies and apoptotic exosome-like vesicles, smaller extracellular vesicles released by endothelial cells downstream of caspase-3 activation. We identified apoptotic exosome-like vesicles as a central trigger for production of anti-perlecan antibodies and acceleration of rejection. Unlike apoptotic bodies, apoptotic exosome-like vesicles triggered the production of anti-perlecan antibodies in naïve mice and enhanced anti-perlecan antibody production and allograft inflammation in mice transplanted with an MHC (major histocompatibility complex)-incompatible aortic graft. The 20S proteasome core was active within apoptotic exosome-like vesicles and controlled their immunogenic activity. Finally, we showed that proteasome activity in circulating exosome-like vesicles increased after vascular injury in mice. These findings open new avenues for predicting and controlling maladaptive humoral responses to apoptotic cell components that enhance the risk of rejection after transplantation.

Objective. Interleukin-1␤ (IL-1␤) and tumor necrosis factor ␣ (TNF␣) stimulate chondrocyte matrix catabolic responses, thereby compromising cartilage homeostasis in osteoarthritis (OA). AMP-activated protein kinase (AMPK), which regulates energy homeostasis and cellular metabolism, also exerts antiinflammatory effects in multiple tissues. This study was undertaken to test the hypothesis that AMPK activity limits chondrocyte matrix catabolic responses to IL-1␤ and TNF␣.Methods. Expression of AMPK subunits was examined, and AMPK␣ activity was ascertained by the phosphorylation status of AMPK␣ Thr 172 in human knee articular chondrocytes and cartilage by Western blotting and immunohistochemistry, respectively. Procatabolic responses to IL-1␤ and TNF␣, such as release of glycosaminoglycan, nitric oxide, and matrix metalloproteinases 3 and 13 were determined by dimethylmethylene blue assay, Griess reaction, and Western blotting, respectively, in cartilage explants and chondrocytes with and without knockdown of AMPK␣ by small interfering RNA.Results. Normal human knee articular chondrocytes expressed AMPK␣1, ␣2, ␤1, ␤2, and ␥1 subunits. Interleukin-1␤ (IL-1␤), tumor necrosis factor ␣ (TNF␣), and certain other proinflammatory cytokines stimulate chondrocyte responses that promote catabolism of type II collagen and proteoglycans (PGs), thereby compromising cartilage extracellular matrix integrity and tissue homeostasis in osteoarthritis (OA) and inflammatory arthritides (1,2). For example, IL-1␤ and TNF␣ induce expression of matrix metalloproteinase 3 (MMP-3) and MMP-13, induce activation of aggrecanases including ADAMTS-5, and stimulate inducible nitric oxide synthase expression and the generation of nitric oxide (NO), a suppressor of PG synthesis (1-3).Recently, the serine/threonine protein kinase AMP-activated protein kinase (AMPK) was observed to exert antiinflammatory effects in tissues other than cartilage, mediated in part by suppression of NF-B activation (4-10). AMPK is a "super-regulator" of

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Stem cell therapy modulates not only the local microenvironment of the brain but also the systemic immune responses. We explored the impact of human multipotent adult progenitor cells (MAPC) modulating splenic activation and peripheral immune responses after ischemic stroke. Hundred twenty-six Long-Evans adult male rats underwent middle cerebral artery occlusion. Twenty-four hours later, they received IV MAPC or saline treatment. At 3 days after infusion, RNA was isolated from the injured cortex and spleen for microarray analysis. Spleen mass, splenocyte phenotype, and releasing cytokines were measured. Serum cytokines, MAPC biodistribution, brain lesion sizes and neurofunctional deficits were compared in rats treated with MAPC or saline with and without spleens. Stroked animals treated with MAPC exhibited genes that more closely resembled animals with sham surgery. Gene categories downregulated by MAPC included leukocyte activation, antigen presentation, and immune effector processing, associated with the signaling pathways regulated by TNF-α, IL-1β, IL-6, and IFN-γ within the brain. MAPC treatment restored spleen mass reduction caused by stroke, elevated Treg cells within the spleen, increased IL-10 and decreased IL-1β released by splenocytes. MAPC reduced IL-6 and IL-1β and upregulated IL-10 serum levels. Compared with saline, MAPC enhance stroke recovery in rats with intact spleens but had no effects in rats without spleens. MAPC restores expression of multiple genes and pathways involved in immune and inflammatory responses after stroke. Immunomodulation of the splenic response by the intravenous administration of MAPC may create a more favorable environment for brain repair after stroke. Stem Cells 2017;35:1290-1302.