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The functional dynamics and cellular sources of oxidative stress are central to understanding MS pathogenesis but remain elusive, due to the lack of appropriate detection methods. Here we employ NAD(P)H fluorescence lifetime imaging to detect functional NADPH oxidases (NOX enzymes) in vivo to identify inflammatory monocytes, activated microglia, and astrocytes expressing NOX1 as major cellular sources of oxidative stress in the central nervous system of mice affected by experimental autoimmune encephalomyelitis (EAE). This directly affects neuronal function in vivo, indicated by sustained elevated neuronal calcium. The systemic involvement of oxidative stress is mirrored by overactivation of NOX enzymes in peripheral CD11b+ cells in later phases of both MS and EAE. This effect is antagonized by systemic intake of the NOX inhibitor and anti-oxidant epigallocatechin-3-gallate. Together, this persistent hyper-activation of oxidative enzymes suggests an “oxidative stress memory” both in the periphery and CNS compartments, in chronic neuroinflammation.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-015-1497-x) contains supplementary material, which is available to authorized users.

Repeatedly activated T helper 1 (Th1) cells present during chronic inflammation can efficiently adapt to the inflammatory milieu, for example, by expressing the transcription factor Twist1, which limits the immunopathology caused by Th1 cells. Here, we show that in repeatedly activated murine Th1 cells, Twist1 and T-bet induce expression of microRNA-148a (miR-148a). miR-148a regulates expression of the proapoptotic gene Bim, resulting in a decreased Bim/Bcl2 ratio. Inhibition of miR-148a by antagomirs in repeatedly activated Th1 cells increases the expression of Bim, leading to enhanced apoptosis. Knockdown of Bim expression by siRNA in miR-148a antagomir-treated cells restores viability of the Th1 cells, demonstrating that miR-148a controls survival by regulating Bim expression. Thus, Twist1 and T-bet not only control the differentiation and function of Th1 cells, but also their persistence in chronic inflammation.

To inhibit binding of the influenza A virus to the host cell glycocalyx, we generate multivalent peptide–polymer nanoparticles binding with nanomolar affinity to the virus via its spike protein hemagglutinin. The chosen dendritic polyglycerol scaffolds are highly biocompatible and well suited for a multivalent presentation. We could demonstrate in vitro that by increasing the size of the polymer scaffold and adjusting the peptide density, viral infection is drastically reduced. Such a peptide–polymer conjugate qualified also in an in vivo infection scenario. With this study we introduce the first non‐carbohydrate‐based, covalently linked, multivalent virus inhibitor in the nano‐ to picomolar range by ensuring low peptide‐ligand density on a larger dendritic scaffold.

The bone marrow maintains memory CD4 T cells, which provide memory to systemic antigens. Here we demonstrate that memory CD4 T cells are reactivated by antigen in the bone marrow. In a secondary immune response, antigen-specific T cells of the bone marrow mobilize and aggregate in immune clusters together with MHC class II-expressing cells, mostly B lymphocytes. They proliferate vigorously and express effector cytokines, but they do not develop into follicular T-helper cells. Neither do the B lymphocytes develop into germinal center B cells in the bone marrow. Within 10 days, the immune clusters disappear again. Within 30 days, the expanded antigen-specific memory CD4 T cells return to memory niches and are maintained again individually as resting cells. Thus, in secondary immune responses in the bone marrow T-cell memory is amplified, while in germinal center reactions of secondary lymphoid organs humoral memory is adapted by affinity maturation.bone marrow | tissue-resident CD4 memory T cells | reactivation | immune clusters | secondary immune reaction I t is increasingly recognized that immunological memory is compartmentalized to different areas of the body, with prominent populations of resident memory T cells being located in the bone marrow (1-5) and epithelial tissues such as the skin (6-8), the lung (9-11), and gastrointestinal (12, 13) and reproductive tracts (14, 15). In the bone marrow (BM), CD4 memory T cells specific for systemic antigens are maintained, even when they are absent from spleen and lymph nodes in mice (1), or the blood in humans (4), arguing that these cells are bona fide residents of the BM. BM resident CD4 memory T cells have been shown to provide efficient cognate help for antibody class switching and affinity maturation of antibodies to B lymphocytes in secondary lymphoid organs upon adoptive transfer (1). In the memory phase of an immune response, such memory T cells rest in the BM in terms of proliferation and activation (1, 4) and are maintained individually in "memory niches" organized by mesenchymal stromal cells (5). Here we analyze the reaction of BM resident CD4 memory T cells to antigen. We show that upon rechallenge antigen-specific CD4 memory T cells proliferate within the BM, independently of immigrating cells, and express effector cytokines. The activated T cells migrate to and gather in immune clusters, clustering with MHC class II-expressing cells, mostly mature B lymphocytes. The activated T cells, however, do not show a follicular helper cell phenotype, nor do the B cells express a germinal center phenotype. Within 30 d after reactivation the immune clusters disappear and the amplified antigen-specific CD4 memory T cells rest again in terms of proliferation, individually dispersed throughout the BM. We show here that immunological CD4 memory is amplified within the BM in a nonfollicular fashion in secondary immune reactions. Results CD4 Memory T Cells Expand in the BM Following Antigen Reactivation.C57BL/6 mice were immunized with 100 μg lymphocytic choriomeningit...

Bone marrow (BM) stromal cells are important in the development and maintenance of cells of the immune system. Using single cell RNA sequencing, we here explore the functional and phenotypic heterogeneity of individual transcriptomes of 1167 murine BM mesenchymal stromal cells. These cells exhibit a tremendous heterogeneity of gene expression, which precludes the identification of defined subpopulations. However, according to the expression of 108 genes involved in the communication of stromal cells with hematopoietic cells, we have identified 14 non‐overlapping subpopulations, with distinct cytokine or chemokine gene expression signatures. With respect to the maintenance of subsets of immune memory cells by stromal cells, we identified distinct subpopulations expressing Il7, Il15 and Tnfsf13b. Together, this study provides a comprehensive dissection of the BM stromal heterogeneity at the single cell transcriptome level and provides a basis to understand their lifestyle and their role as organizers of niches for the long‐term maintenance of immune cells.

Background: Not every T helper type 2 (Th2) lymphocyte imprinted to express interleukin-4 (IL-4) does so when activated.Results: Preventing nuclear translocation of the nuclear factor of activated T cells (NFAT) reduces the number of Th2 lymphocytes reexpressing IL-4.Conclusion: NFAT is the limiting factor determining digital IL-4 expression in Th2 lymphocytes.Significance: This might help us to understand the regulation of immunopathology in allergy and asthma.