Katja Schmitz

Lack of ceramide synthase 2 suppresses the development of experimental autoimmune encephalomyelitis by impairing the migratory capacity of neutrophils

Ceramide synthases (CerS) synthesise ceramides of defined acyl chain lengths, which are thought to mediate cellular processes in a chain length-dependent manner. In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), we observed a significant elevation of CerS2 and its products, C24-ceramides, in CD11b(+) cells (monocytes and neutrophils) isolated from blood. This result correlates with the clinical finding that CerS2 mRNA expression and C24-ceramide levels were significantly increased by 2.2- and 1.5-fold, respectively, in white blood cells of MS patients. The increased CerS2 mRNA/C24-ceramide expression in neutrophils/monocytes seems to mediate pro-inflammatory effects, since a specific genetic deletion of CerS2 in blood cells or a total genetic deletion of CerS2 significantly delayed the onset of clinical symptoms, due to a reduced infiltration of immune cells, in particular neutrophils, into the central nervous system. CXCR2 chemokine receptors, expressed on neutrophils, promote the migration of neutrophils into the central nervous system, which is a prerequisite for the recruitment of further immune cells and the inflammatory process that leads to the development of MS. Interestingly, neutrophils isolated from CerS2 null EAE mice, as opposed to WT EAE mice, were characterised by significantly lower CXCR2 receptor mRNA expression resulting in their reduced migratory capacity towards CXCL2. Most importantly, G-CSF-induced CXCR2 expression was significantly reduced in CerS2 null neutrophils and their migratory capacity was significantly impaired. In conclusion, our data strongly indicate that G-CSF-induced CXCR2 expression is regulated in a CerS2-dependent manner and that CerS2 thereby promotes the migration of neutrophils, thus, contributing to inflammation and the development of EAE and MS.

R‐flurbiprofen attenuates experimental autoimmune encephalomyelitis in mice

R‐flurbiprofen is the non‐cyclooxygenase inhibiting R‐enantiomer of the non‐steroidal anti‐inflammatory drug flurbiprofen, which was assessed as a remedy for Alzheimers disease. Because of its anti‐inflammatory, endocannabinoid‐modulating and antioxidative properties, combined with low toxicity, the present study assessed R‐flurbiprofen in experimental autoimmune encephalomyelitis (EAE) models of multiple sclerosis in mice. Oral R‐flurbiprofen prevented and attenuated primary progressive EAE in C57BL6/J mice and relapsing‐remitting EAE in SJL mice, even if the treatment was initiated on or after the first flare of the disease. R‐flurbiprofen reduced immune cell infiltration and microglia activation and inflammation in the spinal cord, brain and optic nerve and attenuated myelin destruction and EAE‐evoked hyperalgesia. R‐flurbiprofen treatment increased CD4+CD25+FoxP3+ regulatory T cells, CTLA4+ inhibitory T cells and interleukin‐10, whereas the EAE‐evoked upregulation of pro‐inflammatory genes in the spinal cord was strongly reduced. The effects were associated with an increase of plasma and cortical endocannabinoids but decreased spinal prostaglandins, the latter likely due to R to S inversion. The promising results suggest potential efficacy of R‐flurbiprofen in human MS, and its low toxicity may justify a clinical trial.

Dichotomy of CCL21 and CXCR3 in nerve injury-evoked and autoimmunity-evoked hyperalgesia

The chemokine CCL21 is released from injured neurons and acts as a ligand of the chemokine receptor, CXCR3, which likely contributes to pro-inflammatory adaptations and secondary neuronal damage. CCL21-CXCR3 signalling may therefore impact on the development of neuropathic pain. By using the respective knockout mice we show that deficiency of CCL19/21 in plt/plt mice attenuates nerve injury evoked pain but not the hyperalgesia evoked by autoimmune encephalomyelitis (EAE). Oppositely, CXCR3-deficiency had no protective effect after traumatic nerve injury but reduced EAE-evoked hyperalgesia and was associated with reduced clinical EAE scores, a reduction of the pro-inflammatory cell infiltration and reduced upregulation of interferon gamma and interleukin-17 in the spinal cord. In contrast, microglia activation in the spinal cord after traumatic sciatic nerve injury was neither attenuated in CXCR3(-/-) nor plt/plt mice, nor in double knockouts. However, the severity of EAE, but not the hyperalgesia, was also reduced in plt/plt mice, which was associated with reduced infiltration of the spinal cord with CCR7+ T-cells, an increase of CD25+ T-cells and reduced upregulation of CXCL9 and 10, CCL11 and 12. The data show that CCL21 and CXCR3 have dichotomous functions in traumatic and EAE-evoked neuropathic pain suggesting diverse mechanisms likely requiring diverse treatments although both types of neuropathic pain are mediated in part through the immune activation.

Progranulin protects against exaggerated axonal injury and astrogliosis following traumatic brain injury

In response to traumatic brain injury (TBI) microglia/macrophages and astrocytes release inflammatory mediators with dual effects on secondary brain damage progression. The neurotrophic and anti‐inflammatory glycoprotein progranulin (PGRN) attenuates neuronal damage and microglia/macrophage activation in brain injury but mechanisms are still elusive. Here, we studied histopathology, neurology and gene expression of inflammatory markers in PGRN‐deficient mice (Grn−/−) 24 h and 5 days after experimental TBI. Grn−/− mice displayed increased perilesional axonal injury even though the overall brain tissue loss and neurological consequences were similar to wild‐type mice. Brain inflammation was elevated in Grn−/− mice as reflected by increased transcription of pro‐inflammatory cytokines TNFα, IL‐1β, IL‐6, and decreased transcription of the anti‐inflammatory cytokine IL‐10. However, numbers of Iba1+ microglia/macrophages and immigrated CD45+ leukocytes were similar at perilesional sites while determination of IgG extravasation suggested stronger impairment of blood brain barrier integrity in Grn−/− compared to wild‐type mice. Most strikingly, Grn−/− mice displayed exaggerated astrogliosis 5 days after TBI as demonstrated by anti‐GFAP immunohistochemistry and immunoblot. GFAP+ astrocytes at perilesional sites were immunolabelled for iNOS and TNFα suggesting that pro‐inflammatory activation of astrocytes was attenuated by PGRN. Accordingly, recombinant PGRN (rPGRN) attenuated LPS‐ and cytokine‐evoked iNOS and TNFα mRNA expression in cultured astrocytes. Moreover, intracerebroventricular administration of rPGRN immediately before trauma reduced brain damage and neurological deficits, and restored normal levels of cytokine transcription, axonal injury and astrogliosis 5 days after TBI in Grn−/− mice. Our results show that endogenous and recombinant PGRN limit axonal injury and astrogliosis and suggest therapeutic potential of PGRN in TBI. GLIA 2017;65:278–292

Lack of effect of a P2Y6 receptor antagonist on neuropathic pain behavior in mice

Accumulating evidence indicates that various subtypes of purinergic receptors (P2X and P2Y receptor families) play an essential role in the development and the maintenance of neuropathic pain. However, there is only limited data available about the role of P2Y6 receptors in pain processing. Here we detected P2Y6 receptor immunoreactivity in primary afferent neurons of mice and observed an upregulation in response to peripheral nerve injury. However, systemic and intrathecal administration of the P2Y6 receptor antagonist MRS2578 failed to affect the injury-induced neuropathic pain behavior. Our results suggest that P2Y6 receptors, in contrast to other purinergic receptor subtypes, are not critically involved in nerve injury-induced neuropathic pain processing in mice.

Monoclonal Antibodies in Preclinical EAE Models of Multiple Sclerosis: A Systematic Review

Monoclonal antibodies (mAb) are promising therapeutics in multiple sclerosis and multiple new candidates have been developed, hence increasing the need for some agreement for preclinical mAb studies. We systematically analyzed publications of experimental autoimmune encephalomyelitis (EAE) studies showing effects of monoclonal antibodies. A PubMed search retrieved 570 records, out of which 122 studies with 253 experiments were eligible based on experimental design, number of animals and presentation of time courses of EAE scores. Analysis of EAE models, treatment schedules, single and total doses, routes of administration, and onset of treatment from pre-immunization up to 35 days after immunization revealed high heterogeneity. Total doses ranged from 0.1 to 360 mg/kg for observation times of up to 35 days after immunization. About half of experiments (142/253) used total doses of 10–70 mg/kg. Employing this range, we tested anti-Itga4 as a reference mAb at varying schedules and got no, mild or substantial EAE-score reductions, depending on the mouse strain and onset of the treatment. The result agrees with the range of outcomes achieved in 10 reported anti-Itga4 experiments. Studies comparing low and high doses of various mAbs or early vs. late onset of treatment did not reveal dose-effect or timing-effect associations, with a tendency towards better outcomes with preventive treatments starting within the first week after immunization. The systematic comparison allows for extraction of some “common” design characteristics, which may be helpful to further assess the efficacy of mAbs and role of specific targets in preclinical models of multiple sclerosis.

Bioluminescence and Near-infrared Imaging of Optic Neuritis and Brain Inflammation in the EAE Model of Multiple Sclerosis in Mice

Experimental autoimmune encephalomyelitis (EAE) in SJL/J mice is a model for relapsing-remitting multiple sclerosis (RRMS). Clinical EAE scores describing motor function deficits are basic readouts of the immune-mediated inflammation of the spinal cord. However, scores and body weight do not allow for an in vivo assessment of brain inflammation and optic neuritis. The latter is an early and frequent manifestation in about 2/3 of MS patients. Here, we show methods for bioluminescence and near-infrared live imaging to assess EAE evoked optic neuritis, brain inflammation, and blood-brain barrier (BBB) disruption in living mice using an in vivo imaging system. A bioluminescent substrate activated by oxidases primarily showed optic neuritis. The signal was specific and allowed the visualization of medication effects and disease time courses, which paralleled the clinical scores. Pegylated fluorescent nanoparticles that remained within the vasculature for extended periods of time were used to assess the BBB integrity. Near-infrared imaging revealed a BBB leak at the peak of the disease. The signal was the strongest around the eyes. A near-infrared substrate for matrix metalloproteinases was used to assess EAE-evoked inflammation. Auto-fluorescence interfered with the signal, requiring spectral unmixing for quantification. Overall, bioluminescence imaging was a reliable method to assess EAE-associated optic neuritis and medication effects and was superior to the near-infrared techniques in terms of signal specificity, robustness, ease of quantification, and cost.

Treatment with FTY-720 reverses social recognition deficits without affecting clinical scores or impaired motor performance in EAE in SJL mice

Objective: Recently we have developed a reliable and reproducible preclinical chronic-EAN model in the Lewis rat that may prove useful for translational drug studies for CIDP. We have clearly shown that it is a T-cell mediated disease with an accumulation of IL-17 cells and macrophages in its late chronic phase. Since pathophysiological mechanisms involved in CIDP are believed to involve not only cellular but also humoral immunity, we therefore investigated the humoral response in our chronic-EAN model induced in the Lewis rat after injection of thiopalmitoylated P0(180–199) peptide in comparison to the classical acute EAN model induced with P0(180–199). Methods: We investigated by ELISA (at 18, 31, 43 and 57 dpi) in the sera of EAN and chronic-EAN rats the levels of antibodies directed against peptide P0(180–199), the inducing antigen. The reactivity of the antibodies obtained was tested on sciatic nerve by immunohistochemistry. To check the possibility of an epitope spreading at the late phase of the chronic disease, the reactivity of the sera at 60 dpi was tested by ELISA against different peptides described as neuritogenic: P0(56–71), P0(152–171), P0(180–199) and P2(57– 81). We also investigated the presence of B-cells in the sciatic nerve by immunohistochemistry using a mouse anti-rat CD45RA MnAb. Results: High levels of antibodies against P0(180–199) were found in chronic-EAN and EAN rats, but the antibody reactivity remained high in the chronic group for the duration of the disease, whereas it started to decline in the EAN group by day 57. At the late phase of the chronic disease (60 dpi) we found in the sera significant levels of antibodies against peptide P0(152–171) but this was not statistically different from the EAN group. No reactivity was detected against the other peptides. The rat polyclonal anti P0(180–199) antibodies labeled nicely the PNS myelin in a control sciatic nerve, highly comparable to the myelin labeling with the commercial anti-P0 MnAb. This indicates that the anti-P0(180–199) is able to recognize the corresponding peptide sequence when protein P0 is inserted in the myelin membrane. The presence of B cells was detected in the sciatic nerve of EAN rats and also of chronic-EAN rats but in greater number. Conclusion: We have indeed shown that there is a humoral response in our chronic model, but further studies are needed to clarify the roles of the antibodies and B-cells in the persistence of the chronicity of the disease.

R-flurbiprofen attenuates experimental autoimmune encephalomyelitis in mice

compared with that of control siRNA. Collagen I, III, IV, V and decorin expression were found in the perivascular space and in the parenchyma. Collagen I, IV and V are expressed by astrocytes which were also expressing RGC-32. Since RGC-32was found to be involved inmediation of TGF-beta effects, we investigated its role in TGF-beta-induced ECM expression and reactive astrocyte marker α-smooth muscle actin (αSMA). In cultured astrocytes, α-SMA, collagen I, IV and V as well as fibronectin were significantly induced at 18 h of stimulation with TGFbeta. Next, we silenced RGC-32 expression by transfecting astrocytes with siRGC-32 and compared the effect of this treatment to that of control siRNA. We found that α-SMA expression was significantly reduced after RGC-32 silencing (p b 0.05). In addition we found that RGC-32 silencing resulted in a significant reduction in TGF-beta-induced collagen I (p b 0.01), collagen IV (p b 0.02), collagen V (p b 0.05) and fibronectin (p b 0.05) expression. Using astrocytes isolated from RGC-32 knockout (KO) mouse we found that TGF-beta-induced collagen IV and alpha-SMA expression were significantly reduced in RGC-32 KO when compared with wild type mouse. The effect of RGC-32 silencing on alpha-SMAexpression suggests that RGC-32 is required for the transition of astrocytes to a reactive state. Our data also indicate that RGC-32 plays an important role in the TGF-beta-mediated induction of ECMexpression in astrocytes. RGC-32 may therefore represent a useful new target for therapeutic intervention in MS.