Lymphocyte homing into the intestine is mediated by binding of leukocytes to mucosal addressin cell adhesion molecule 1 (MAdCAM-1), expressed on endothelial cells. Currently, the immune system of the gut is considered a major modulator not only of inflammatory bowel disease, but also of extra-intestinal autoimmune disorders, including multiple sclerosis (MS). Despite intense research in this field, the exact role of the intestine in the pathogenesis of (neuro-)inflammatory disease conditions remains to be clarified. This prompted us to investigate the role of MAdCAM-1 in immunological processes in the intestine during T cell-mediated autoimmunity of the central nervous system (CNS). Using the experimental autoimmune encephalomyelitis model of MS, we show that MAdCAM-1-deficient (MAdCAM-1-KO) mice are less susceptible to actively MOG35−55-induced disease. Protection from disease was accompanied by decreased numbers of immune cells in the lamina propria and Peyer's patches as well as reduced immune cell infiltration into the spinal cord. MOG35−55-recall responses were intact in other secondary lymphoid organs of MAdCAM-1-KO mice. The composition of specific bacterial groups within the microbiome did not differ between MAdCAM-1-KO mice and controls, while MAdCAM-1-deficiency severely impaired migration of MOG35−55-activated lymphocytes to the gut. Our data indicate a critical role of MAdCAM-1 in the development of CNS inflammation by regulating lymphocyte homing to the intestine, and may suggest a role for the intestinal tract in educating lymphocytes to become encephalitogenic.

ObjectiveImmunological studies have demonstrated a plethora of beneficial effects of dimethyl fumarate (DMF) on various cell types. However, the cellular and molecular targets are incompletely understood and response markers are scarce. Here, we focus on the relation between nuclear factor (erythroid-derived 2)-like 2 (NRF2) pathway induction under DMF therapy and the composition of the blood immune cell compartment and clinical efficacy in relapsing-remitting multiple sclerosis (MS) patients. MethodsWe explored effects of DMF on peripheral immune cell subsets by flow cytometric and transcriptional analysis of serial blood samples obtained from 43 MS patients during the first year of therapy. ResultsGene expression analysis proved activation of NRF2 signaling under DMF therapy that was paralleled by a temporal expansion of FoxP3(+) regulatory T cells, CD56(bright) natural killer cells, plasmacytoid dendritic cells, and a decrease in CD8(+) T cells, B cells, and type 1 myeloid dendritic cells. In a subgroup of 28 patients with completely available clinical data, individuals with higher levels of the NRF2 target gene NAD(P)H quinone dehydrogenase 1 (NQO1) 4-6 weeks after DMF therapy initiation were more likely to achieve no evidence of disease activity status 1 year later. The degree of NQO1 induction further correlated with patient age. InterpretationWe demonstrate that positive effects of DMF on the clinical outcome are paralleled by induction of the antioxidant NRF2 transcriptional pathway and a shift toward regulatory immune cell subsets in the periphery. Our data identify a role of the NRF2 pathway as potential biomarker for DMF treatment in MS.

Macrophages are essential players of the innate immune system which are involved in the initiation and progression of various inflammatory and autoimmune diseases including neuroinflammation. In the past few years, it has become increasingly clear that the regulation of macrophage responses by the local tissue milieu is also influenced by mediators which were first discovered as regulators in the nervous or also cardiovascular system. Here, the renin-angiotensin system (RAS) is a major focus of current research. Besides its classical role in blood pressure control, body fluid, and electrolyte homeostasis, the RAS may influence (auto)immune responses, modulate T cells, and particularly act on macrophages via different signaling pathways. Activation of classical RAS pathways including angiotensin (Ang) II and AngII type 1 (AT 1 R) receptors may drive pro-inflammatory macrophage responses in neuroinflammation via regulation of chemokines. More recently, alternative RAS pathways were described, such as binding of Ang-(1–7) to its receptor Mas. Signaling via Mas pathways may counteract some of the AngII/AT 1 R-mediated effects. In macrophages, the Ang-(1–7)/Mas exerts beneficial effects on neuroinflammation via modulating macrophage polarization, migration, and T cell activation in vitro and in vivo. These data delineate a pivotal role of the RAS in inflammation of the nervous system and identify RAS modulation as a potential new target for immunotherapy with a special focus on macrophages.

To date, the intracellular signaling pathways involved in dendritic cell (DC) function are poorly understood. The antioxidative transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) has been shown to affect maturation, function, and subsequent DC-mediated T cell responses of murine and human DCs. In experimental autoimmune encephalomyelitis (EAE), as prototype animal model for a T helper cell-mediated autoimmune disease, antigen presentation, cytokine production, and costimulation by DCs play a major role. We explore the role of Nrf2 in DC function, and DC-mediated T cell responses during T cell-mediated autoimmunity of the central nervous system using genetic ablation and pharmacological activation in mice and men to corroborate our data in a translational setting. In murine and human DCs, monomethyl fumarate induced Nrf2 signaling inhibits DC maturation and DC-mediated T cell proliferation by reducing inflammatory cytokine production and expression of costimulatory molecules. In contrast, Nrf2-deficient DCs generate more activated T helper cells (Th1/Th17) but fewer regulatory T cells and foster T cell proliferation. Transfer of DCs with Nrf2 activation during active EAE reduces disease severity and T cell infiltration. Our data demonstrate that Nrf2 signaling modulates autoimmunity in murine and human systems via inhibiting DC maturation and function thus shedding further light on the mechanism of action of antioxidative stress pathways in antigen-presenting cells.

There has been a marked increase in the incidence of autoimmune diseases like multiple sclerosis (MS) in the last decades which is most likely driven by a change in environmental factors. Here, growing evidence suggests that ingredients of a Western diet like high intake of sodium chloride (NaCl) or saturated fatty acids may impact systemic immune responses, thus increasing disease susceptibility. Recently, we have shown that high dietary salt or long-chain fatty acid (LCFA) intake indeed aggravates T helper (Th) cell responses and neuroinflammation.

Growing empirical evidence suggests that nutrition and bacterial metabolites might impact the systemic immune response in the context of disease and autoimmunity. We report that long-chain fatty acids (LCFAs) enhanced differentiation and proliferation of T helper 1 (Th1) and/or Th17 cells and impaired their intestinal sequestration via p38-MAPK pathway. Alternatively, dietary short-chain FAs (SCFAs) expanded gut T regulatory (Treg) cells by suppression of the JNK1 and p38 pathway. We used experimental autoimmune encephalomyelitis (EAE) as a model of T cell-mediated autoimmunity to show that LCFAs consistently decreased SCFAs in the gut and exacerbated disease by expanding pathogenic Th1 and/or Th17 cell populations in the small intestine. Treatment with SCFAs ameliorated EAE and reduced axonal damage via long-lasting imprinting on lamina-propria-derived Treg cells. These data demonstrate a direct dietary impact on intestinal-specific, and subsequently central nervous system-specific, Th cell responses in autoimmunity, and thus might have therapeutic implications for autoimmune diseases such as multiple sclerosis.

Kidney cell death plays a key role in the progression of life-threatening renal diseases, such as acute kidney injury and chronic kidney disease. Injured and dying epithelial and endothelial cells take part in complex communication with the innate immune system, which drives the progression of cell death and the decrease in renal function. To improve our understanding of kidney cell death dynamics and its impact on renal disease, a study approach is needed that facilitates the visualization of renal function and morphology in real time. Intravital multiphoton microscopy of the kidney has been used for more than a decade and made substantial contributions to our understanding of kidney physiology and pathophysiology. It is a unique tool that relates renal structure and function in a time- and spatial-dependent manner. Basic renal function, such as microvascular blood flow regulation and glomerular filtration, can be determined in real time and homeostatic alterations, which are linked inevitably to cell death and can be depicted down to the subcellular level. This review provides an overview of the available techniques to study kidney dysfunction and inflammation in terms of cell death in vivo, and addresses how this novel approach can be used to improve our understanding of cell death dynamics in renal disease.

Recently, an alternative renin-angiotensin system pathway has been described, which involves binding of angiotensin-(1-7) to its receptor Mas. The Mas axis may counterbalance angiotensin-II-mediated proinflammatory effects, likely by affecting macrophage function. Here we investigate the role of Mas in murine models of autoimmune neuroinflammation and atherosclerosis, which both involve macrophage-driven pathomechanisms. Mas signaling affected macrophage polarization, migration, and macrophage-mediated T-cell activation. Mas deficiency exacerbated the course of experimental autoimmune encephalomyelitis and increased macrophage infiltration as well as proinflammatory gene expression in the spleen and spinal cord. Furthermore, Mas deficiency promoted atherosclerosis by affecting macrophage infiltration and migration and led to increased oxidative stress as well as impaired endothelial function in ApoE-deficient mice. In summary, we identified the Mas axis as an important factor in macrophage function during inflammation of the central nervous and vascular system in vivo. Modulating the Mas axis may constitute an interesting therapeutic target in multiple sclerosis and/or atherosclerosis.

The basolateral chloride channel CLCKb facilitates Cl reabsorption in the distal nephron. Mutations in clcnkb were associated with Bartter's syndrome type 3 in humans. To address the function of clcnkb for renal salt reabsorption, we generated a mouse line with a null mutation of clcnkb by the use of the Zn finger technology. Clcnkb‐deficient mice were viable and were born in Mendelian ratio. At the age of 4 weeks, clcnkb−/− mice showed a slight growth retardation, with a body weight of 14.0±0.5 g compared with 19.7±0.5 g in WT (p<0.0001). Ambient urine osmolarity was markedly reduced in Clcnkb‐deficient mice (590±39 vs. 2216±132 mosmol/L in WT; p<0.0001). During water restriction (12 hrs), urinary osmolarity increased to 1633±153 mosmol/L and 3769±129 mosmol/L in clcnkb−/− and WT mice (n=12; p<0.0001), which was accompanied by a loss of body weight of 12±0.4% and 8±0.2%, respectively (p<0.0001). Fractional urinary K + excretion averaged 0.21±0.02 mmol/mosmol (clcnkb−/−) and 0.11±0.01 mmol/mosmol (WT; p=0.0008), suggesting increased K secretion in the collecting duct of clcnkb−/− mice. The salt‐losing phenotype of clcnkb−/− mice was associated with a reduced plasma volume (2.5±0.6% of body weight vs. 3.9±0.2% in WT; p=0.0230), a reduced GFR (1040±97 vs. 1395±129 μl/min/100g bw in WT; n=9; p= 0.0411), and massively increased plasma renin concentrations (35720±8941 vs. 56±8 ng Ang I/ml/min in WT, respectively; n=11; p<0.0001). Our data suggest that clcnkb is crucial for renal salt reabsorption and concentrating ability. Clcnkb‐deficient mice appear as a suitable model of Bartter's syndrome type 3.

We recently showed that angiotensin II (ANGII) acutely increases the albumin filtration in the healthy kidney. Here we used intravital microscopy to assess the effects of ANGII on podocyte function in rats. Acute infusion of 0, 10, 30, 60, and 80 ng/kg/min ANGII enhanced the endocytosis of albumin and resulted in an average of 0, 0, 3.7±2.2, 72.3±18.6 (p=0.0004) and 239.4±34.6 µm 3 (p<0.0001), respectively, of albumin‐containing vesicles/glomerulus. This effect was abolished in the presence of the AT1 receptor antagonist losartan. Immunostainings of ANGII‐infused kidneys confirmed the presence of albumin‐containing vesicles in the podocytes, which colocalized with podocin and the multiligand receptor megalin. Furthermore, in the presence of gentamicin, podocyte endocytosis of albumin was markedly reduced to 1.5±1.5 and 22.5±6.3 µm 3 during infusion of 60 and 80 ng/kg/min ANGII (p=.0025 and .0001 vs. non‐gentamicin‐treated rats, respectively), suggesting a megalin‐dependent uptake mechanism. ANGII infusion increased the concentration of albumin in the subpodocyte space, a potential source for endocytic protein uptake, and this concentration was further increased in the presence of gentamicin. Some of the endocytic vesicles were acidified, and colocalized with the lysosomal marker lysotracker in vivo , suggesting lysosomal degradation. Another subset of vesicles migrated from the capillary to the apical aspect of the podocyte and was eventually shed into the urinary space. In summary, the transcellular transport of proteins across the podocyte constitutes a new pathway of glomerular protein filtration. ANGII enhances the endocytosis and shedding of plasma albumin by podocytes and this may eventually impair podocyte function.

OBJECTIVE: To investigate the impact of diet, in particular fatty acids on the local immune response and T cell differentiation in the gut, under normal conditions and in the context of CNS autoimmunity. BACKGROUND: Recent advances in the field of microbiomics have opened new opportunities for investigating interactions between nutrition, gut microbiome and the local cellular immune response, even in autoimmunity. However, the interaction between the gut microbiome, nutrition and the local immune response is less well understood. DESIGN/METHODS: We analyzed the effect of fatty acids with different chain lengths on differential T cell differentiation ex vivo and in vivo in the small intestine of healthy wild type mice and in an MOG induced experimental model of multiple sclerosis, the experimental autoimmune encephalomyelitis (EAE). RESULTS: We report an opposing effect of dietary short chain fatty acids (SCFA) and the less well studied medium or long chain FA (MCFA/LCFA) on T helper (Th) cells:MCFA and LCFA promote differentiation of Th1 and Th17 cells and lead to an impaired sequestration of intestinal Th1 and Th17 cells via p38 MAPK. In contrast, dietary SCFA enhance regulatory T cell (Treg) differentiation and expand gut Treg by lipin-2/JIP2 dependent suppression of the JNK1 pathway. In a model of T cell mediated autoimmunity, LCFA and MCFA exacerbate EAE and increase pathogenic T cell populations by skewing the microbiome composition towards a pro-inflammatory Th1/Th17 inducing phenotype. On the contrary, treatment with SCFA ameliorates EAE and reduces axonal damage via long-lasting imprinting on lamina propria derived CD4+ CD25+ Foxp3+ Treg. CONCLUSIONS: Our results suggest a direct dietary impact on intestinal and subsequently CNS specific Th cell responses in extra-intestinal autoimmunity and may have therapeutic implications for autoimmune diseases like multiple sclerosis. Disclosure: Dr. Haghikia has nothing to disclose. Dr. Jorg has nothing to disclose. Dr. Alexander has nothing to disclose. Dr. Berg has nothing to disclose. Dr. Hammer has nothing to disclose. Dr. Akkad has nothing to disclose. Dr. Muller has nothing to disclose. Dr. Gold has received personal compensation for activities with Bayer HealthCare, Biogen Idec, Merck Serono, Novartis, and Teva Neuroscience. Dr. Linker has received personal compensation for activities with Bayer Pharmaceuticals, Inc., Biogen Idec, Merck Serono, Novartis, Sanofi-Aventis Pharmaceuticals, and Teva Neuroscience.

Neuroprotective approaches for central nervous system regeneration have not been successful in clinical practice so far and compounds that enhance remyelination are still not available for patients with multiple sclerosis. The objective of this study was to determine potential regenerative effects of the substance cytidine-5'-diphospho (CDP)-choline in two different murine animal models of multiple sclerosis. The effects of exogenously applied CDP-choline were tested in murine myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis. In addition, the cuprizone-induced mouse model of de- and remyelination was used to specifically test the hypothesis that CDP-choline directly increases remyelination. We found that CDP-choline ameliorated the disease course of experimental autoimmune encephalomyelitis and exerted beneficial effects on myelin, oligodendrocytes and axons. After cuprizone-induced demyelination, CDP-choline effectively enhanced myelin regeneration and reversed motor coordination deficits. The increased remyelination arose from an increase in the numbers of proliferating oligodendrocyte precursor cells and oligodendrocytes. Further in vitro studies suggest that this process is regulated by protein kinase C. We thus identified a new mechanism to enhance central nervous system remyelination via the choline pathway. Due to its regenerative action combined with an excellent safety profile, CDP-choline could become a promising substance for patients with multiple sclerosis as an add-on therapy.

OBJECTIVE: To assess the influence of dimethyl fumarate (DMF) on intestinal T helper (Th) cells in relation to central nervous system (CNS) specific immune responses in experimental autoimmune encephalomyelitis (EAE). BACKGROUND: DMF is a novel oral therapeutic option for multiple sclerosis exerting immunomodulatory and possibly also cytoprotective effects via activation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) mediated pathways. Although the small intestine is an important site of immunoregulation in autoimmunity, little is known on the influence of DMF on the gut and the intestinal immune system. DESIGN/METHODS: After immunization with myelin oligodendrocyte glycoprotein in complete Freund´s adjuvants, mice received DMF (15 mg/kg/bid) solved in methocel via oral gavage. Controls were treated with methocel only. Spleen and gut were dissected 3, 7 and 14 days after EAE induction for analysis via flow cytometry or real-time PCR. RESULTS: After 14, but not 7 days of treatment with DMF, mRNA levels of the Nrf2 target genes NAD(P)H-dehydrogenase-quinone-1 and aldo-keto-reductase-family-1-member-B8 were robustly elevated in the duodenum, but not jejunum of naïve and EAE mice (p<0.05). DMF increased the expression of arylhydrocarbon-receptor and transforming- growth-factor-β in the gut of naïve mice but decreased the interferon gamma mRNA abundance under EAE conditions (p<0.05). The frequency of Th1 cells among total lamina propria lymphocytes as well as splenic Th1 cells was decreased under DMF treatment at day 14 of EAE while numbers of Foxp3 positive regulatory T cells were elevated in the small intestine (Th1 cells: 9.8[percnt] ± 1.3[percnt] vs 5.3[percnt] ± 0.46[percnt], Treg cells: 1.2[percnt] ± 0.04[percnt] vs 1.6[percnt] ± 0.05, each p<0.05). CONCLUSIONS: Chronic DMF treatment in EAE modulates intestinal T cell regulation and activates Nrf2 specific pathways in the gut. Further data linking these effects to the microbiome and CNS specific T cell responses will be presented. Study Supported by: BiogenIdec