Isabell Hamann

Frequency of blood CX3CR1-positive natural killer cells correlates with disease activity in multiple sclerosis patients

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) characterized by enormous variability in its clinical presentation and course, and for which clear diagnostic parameters are lacking. Here we performed an RNA screen in peripheral mononuclear cells from relapsing‐remitting (RR) and primary progressive (PP) MS patients compared with healthy donors (HD) that indicated, among other findings, a role for the chemokine receptor CX3CR1 as a diagnostic marker. Gene expression and flow cytometric analyses demonstrated a significantly lower expression of CX3CR1 in MS patients compared with healthy individuals. The subpopulation of cells responsible for causing this reduced expression of CX3CR1 consisted exclusively of natural killer (NK) cells. Importantly, we found a correlation between disease activity and frequency of CX3CR1‐positive NK cells in RRMS patients. These findings emphasize the role of NK cells in the development and course of MS and provide evidence for CX3CR1 expression as a marker for MS patients and disease activity.

Magnetic resonance elastography reveals altered brain viscoelasticity in experimental autoimmune encephalomyelitis

Cerebral magnetic resonance elastography (MRE) measures the viscoelastic properties of brain tissues in vivo. It was recently shown that brain viscoelasticity is reduced in patients with multiple sclerosis (MS), highlighting the potential of cerebral MRE to detect tissue pathology during neuroinflammation. To further investigate the relationship between inflammation and brain viscoelasticity, we applied MRE to a mouse model of MS, experimental autoimmune encephalomyelitis (EAE). EAE was induced and monitored by MRE in a 7-tesla animal MRI scanner over 4 weeks. At the peak of the disease (day 14 after immunization), we detected a significant decrease in both the storage modulus (G′) and the loss modulus (G″), indicating that both the elasticity and the viscosity of the brain are reduced during acute inflammation. Interestingly, these parameters normalized at a later time point (day 28) corresponding to the clinical recovery phase. Consistent with this, we observed a clear correlation between viscoelastic tissue alteration and the magnitude of perivascular T cell infiltration at both day 14 and day 28. Hence, acute neuroinflammation is associated with reduced mechanical cohesion of brain tissues. Moreover, the reduction of brain viscoelasticity appears to be a reversible process, which is restored when inflammation resolves. For the first time, our study has demonstrated the applicability of cerebral MRE in EAE, and showed that this novel imaging technology is highly sensitive to early tissue alterations resulting from the inflammatory processes. Thus, MRE may serve to monitor early stages of perivascular immune infiltration during neuroinflammation.

Therapeutic targeting of chemokine signaling in Multiple Sclerosis

Multiple Sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) that is initiated and maintained by continuous migration of inflammatory immune cells from the periphery into the target organ. However, in autoimmunity, migration of immune cells is not only involved in the pathogenesis but also in the down-modulation of the autoimmune attack, which is probably mediated by the infiltration of certain regulatory immune cell populations inside the affected organs. The migratory activity of both proinflammatory and regulatory leucocytes is controlled by chemokines and their receptors. Thus, targeting the directed migration of immune cells and regulating leukocyte trafficking across the blood-brain barrier (BBB) by means of modulation of chemokine signaling receptors might open up new therapeutic avenues not only for MS but also for other autoimmune diseases. In this review we summarize the chemotactic signaling pathways known to be involved in neuroinflammation to date and the viability of these pathways as targets for therapeutic strategies.

Analyses of phenotypic and functional characteristics of CX3CR1-expressing natural killer cells.

Summary We previously demonstrated a correlation between the frequency of CX3CR1‐expressing human natural killer (NK) cells and disease activity in multiple sclerosis and showed that CX3CR1high NK cells were more cytotoxic than their CX3CR1neg/low counterparts. Here we aimed to determine whether human NK cell fractions defined by CX3CR1 represent distinct subtypes. Phenotypic and functional NK cell analyses revealed that, distinct from CX3CR1high, CX3CR1neg/low NK cells expressed high amounts of type 2 cytokines, proliferated robustly in response to interleukin‐2 and promoted a strong up‐regulation of the key co‐stimulatory molecule CD40 on monocytes. Co‐expression analyses of CX3CR1 and CD56 demonstrated the existence of different NK cell fractions based on the surface expression of these two surface markers, the CX3CR1neg CD56bright, CX3CR1neg CD56dim and CX3CR1high CD56dim fractions. Additional investigations on the expression of NK cell receptors (KIR, NKG2A, NKp30 and NKp46) and the maturation markers CD27, CD62L and CD57 indicated that CX3CR1 expression of CD56dim discriminated between an intermediary CX3CR1neg CD56dim and fully mature CX3CR1high CD56dim NK cell fractions. Hence, CX3CR1 emerges as an additional differentiation marker that may link NK cell maturation with the ability to migrate to different organs including the central nervous system.

Arteriogenesis Is Modulated By Bradykinin Receptor Signaling

Rationale: Positive outward remodeling of pre-existing collateral arteries into functional conductance arteries, arteriogenesis, is a major endogenous rescue mechanism to prevent cardiovascular ischemia. Collateral arterial growth is accompanied by expression of kinin precursor. However, the role of kinin signaling via the kinin receptors (B1R and B2R) in arteriogenesis is unclear. Objective: The purpose of this study was to elucidate the functional role and mechanism of bradykinin receptor signaling in arteriogenesis. Methods and Results: Bradykinin receptors positively affected arteriogenesis, with the contribution of B1R being more pronounced than B2R. In mice, arteriogenesis upon femoral artery occlusion was significantly reduced in B1R mutant mice as evidenced by reduced microspheres and laser Doppler flow perfusion measurements. Transplantation of wild-type bone marrow cells into irradiated B1R mutant mice restored arteriogenesis, whereas bone marrow chimeric mice generated by reconstituting wild-type mice with B1R mutant bone marrow showed reduced arteriogenesis after femoral artery occlusion. In the rat brain 3-vessel occlusion arteriogenesis model, pharmacological blockade of B1R inhibited arteriogenesis and stimulation of B1R enhanced arteriogenesis. In the rat, femoral artery ligation combined with arterial venous shunt model resulted in flow-driven arteriogenesis, and treatment with B1R antagonist R715 decreased vascular remodeling and leukocyte invasion (monocytes) into the perivascular tissue. In monocyte migration assays, in vitro B1R agonists enhanced migration of monocytes. Conclusions: Kinin receptors act as positive modulators of arteriogenesis in mice and rats. B1R can be blocked or therapeutically stimulated by B1R antagonists or agonists, respectively, involving a contribution of peripheral immune cells (monocytes) linking hemodynamic conditions with inflammatory pathways.

Characterization of natural killer cells in paired CSF and blood samples during neuroinflammation

Natural killer (NK) cells from paired CSF and blood samples of patients with multiple sclerosis (MS), other neuroinflammatory diseases (IND), and non-inflammatory neurological diseases (NIND) were characterized using flow cytometry. NK cell frequency in CSF was overall decreased compared to blood, particularly in MS patients. In contrast to blood NK cells, during neuroinflammation, CSF NK cells display an immature phenotype with bright expression of CD56 and CD27 and reduced CX3CR1 expression. Our findings suggest that, as for central memory T cells, CSF may represent an intermediary compartment for NK cell trafficking and differentiation before entering the CNS parenchyma.

Mitoxantrone Induces Natural Killer Cell Maturation in Patients with Secondary Progressive Multiple Sclerosis

Mitoxantrone is one of the few drugs approved for the treatment of progressive multiple sclerosis (MS). However, the prolonged use of this potent immunosuppressive agent is limited by the appearance of severe side effects. Apart from its general cytotoxic effect, the mode of action of mitoxantrone on the immune system is poorly understood. Thus, to develop safe therapeutic approaches for patients with progressive MS, it is essential to elucidate how mitoxantrone exerts it benefits. Accordingly, we initiated a prospective single-arm open-label study with 19 secondary progressive MS patients. We investigated long-term effects of mitoxantrone on patient peripheral immune subsets using flow cytometry. While we corroborate that mitoxantrone persistently suppresses B cells in vivo, we show for the first time that treatment led to an enrichment of neutrophils and immunomodulatory CD8low T cells. Moreover, sustained mitoxantrone applications promoted not only persistent NK cell enrichment but also NK cell maturation. Importantly, this mitoxantrone-induced NK cell maturation was seen only in patients that showed a clinical response to treatment. Our data emphasize the complex immunomodulatory role of mitoxantrone, which may account for its benefit in MS. In particular, these results highlight the contribution of NK cells to mitoxantrone efficacy in progressive MS.

CX3CR1-dependent recruitment of mature NK cells into the central nervous system contributes to control autoimmune neuroinflammation

Fractalkine receptor (CX3CR1)‐deficient mice develop very severe experimental autoimmune encephalomyelitis (EAE), associated with impaired NK cell recruitment into the CNS. Yet, the precise implications of NK cells in autoimmune neuroinflammation remain elusive. Here, we investigated the pattern of NK cell mobilization and the contribution of CX3CR1 to NK cell dynamics in the EAE. We show that in both wild‐type and CX3CR1‐deficient EAE mice, NK cells are mobilized from the periphery and accumulate in the inflamed CNS. However, in CX3CR1‐deficient mice, the infiltrated NK cells displayed an immature phenotype contrasting with the mature infiltrates in WT mice. This shift in the immature/mature CNS ratio contributes to EAE exacerbation in CX3CR1‐deficient mice, since transfer of mature WT NK cells prior to immunization exerted a protective effect and normalized the CNS NK cell ratio. Moreover, mature CD11b+ NK cells show higher degranulation in the presence of autoreactive 2D2 transgenic CD4+ T cells and kill these autoreactive cells more efficiently than the immature CD11b− fraction. Together, these data suggest a protective role of mature NK cells in EAE, possibly through direct modulation of T cells inside the CNS, and demonstrate that mature and immature NK cells are recruited into the CNS by distinct chemotactic signals.

Treatment of Chronic Experimental Autoimmune Encephalomyelitis with Epigallocatechin-3-Gallate and Glatiramer Acetate Alters Expression of Heme-Oxygenase-1

We previously demonstrated that epigallocatechin-3-gallate (EGCG) synergizes with the immunomodulatory agent glatiramer acetate (GA) in eliciting anti-inflammatory and neuroprotective effects in the relapsing-remitting EAE model. Thus, we hypothesized that mice with chronic EAE may also benefit from this combination therapy. We first assessed how a treatment with a single dose of GA together with daily application of EGCG may modulate EAE. Although single therapies with a suboptimal dose of GA or EGCG led to disease amelioration and reduced CNS inflammation, the combination therapy had no effects. While EGCG appeared to preserve axons and myelin, the single GA dose did not improve axonal damage or demyelination. Interestingly, the neuroprotective effect of EGCG was abolished when GA was applied in combination. To elucidate how a single dose of GA may interfere with EGCG, we focused on the anti-inflammatory, iron chelating and anti-oxidant properties of EGCG. Surprisingly, we observed that while EGCG induced a downregulation of the gene expression of heme oxygenase-1 (HO-1) in affected CNS areas, the combined therapy of GA+EGCG seems to promote an increased HO-1 expression. These data suggest that upregulation of HO-1 may contribute to diminish the neuroprotective benefits of EGCG alone in this EAE model. Altogether, our data indicate that neuroprotection by EGCG in chronic EAE may involve regulation of oxidative processes, including downmodulation of HO-1. Further investigation of the re-dox balance in chronic neuroinflammation and in particular functional studies on HO-1 are warranted to understand its role in disease progression.

Preproenkephalin Expression in Peripheral Blood Mononuclear Cells of Acutely Underweight and Recovered Patients with Anorexia Nervosa

Background: The prohormone preproenkephalin (ppE) and its derived peptides are involved in leukocyte functioning as well as in the regulation of hunger and satiety. Various abnormalities of the immune and endocrine systems have been described in states of malnutrition such as anorexia nervosa (AN). We hypothesized that ppE expression in AN patients may vary depending on the state of the disorder and the extent of malnutrition. Methods: Expression of ppE mRNA was analysed in peripheral blood mononuclear cells of 29 underweight and 29 weight-recovered patients with AN and compared to that in 29 healthy control women. The extent of malnutrition was characterized by BMI and plasma leptin. Psychological distress and eating disorder specific-psychopathology was determined with the Symptom Checklist-90-Revised and the Eating Disorders Inventory-2. Results: ppE gene expression was similar in all 3 groups and was not related to nutritional status or eating disorder symptoms. However, a significant negative correlation was found between ppE expression and obsessive-compulsive, depressive and anxious symptoms. In addition, ppE expression was higher in smokers compared to non-smokers. Conclusion:Although malnutrition and hypoleptinaemia as seen in patients with AN were not related to peripheral ppE expression, we demonstrated reduced ppE expression in patients with elevated psychological distress. Similar associations have been shown in animal models of stress. It remains speculative if psychological symptoms and/or stress may augment immune abnormalities in AN patients via a pathway that is independent of nutritional status and involves ppE.