An Alternative Pathway of Imiquimod-Induced Psoriasis-Like Skin Inflammation in the Absence of Interleukin-17 Receptor A Signaling

Topical application of imiquimod (IMQ) on the skin of mice induces inflammation with common features found in psoriatic skin. Recently, it was postulated that IL-17 has an important role both in psoriasis and in the IMQ model. To further investigate the impact of IL-17RA signaling in psoriasis, we generated IL-17 receptor A (IL-17RA)–deficient mice (IL-17RAdel) and challenged these mice with IMQ. Interestingly, the disease was only partially reduced and delayed but not abolished when compared with controls. In the absence of IL-17RA, we found persisting signs of inflammation such as neutrophil and macrophage infiltration within the skin. Surprisingly, already in the naive state, the skin of IL-17RAdel mice contained significantly elevated numbers of Th17- and IL-17-producing γδ T cells, assuming that IL-17RA signaling regulates the population size of Th17 and γδ T cells. Upon IMQ treatment of IL-17RAdel mice, these cells secreted elevated amounts of tumor necrosis factor-α, IL-6, and IL-22, accompanied by increased levels of the chemokine CXCL2, suggesting an alternative pathway of neutrophil and macrophage skin infiltration. Hence, our findings have major implications in the potential long-term treatment of psoriasis by IL-17-targeting drugs

A20 regulates lymphocyte adhesion in murine neuroinflammation by restricting endothelial ICOSL expression in the CNS.

A20 is a ubiquitin-modifying protein that negatively regulates NF-κB signaling. Mutations in A20/TNFAIP3 are associated with a variety of autoimmune diseases, including multiple sclerosis (MS). We found that deletion of A20 in central nervous system (CNS) endothelial cells (ECs) enhances experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. A20∆CNS-EC mice showed increased numbers of CNS-infiltrating immune cells during neuroinflammation and in the steady state. While the integrity of the blood-brain barrier (BBB) was not impaired, we observed a strong activation of CNS-ECs in these mice, with dramatically increased levels of the adhesion molecules ICAM-1 and VCAM-1. We discovered ICOSL as adhesion molecule expressed by A20-deficient CNS-ECs. Silencing of ICOSL in CNS microvascular ECs partly reversed the phenotype of A20∆CNS-EC mice without reaching statistical significance and delayed the onset of EAE symptoms in wildtype mice. In addition, blocking of ICOSL on primary mouse brain microvascular endothelial cells (pMBMECs) impaired the adhesion of T cells in vitro. Taken together, we here propose that CNS EC-ICOSL contributes to the firm adhesion of T cells to the BBB, promoting their entry into the CNS and eventually driving neuroinflammation

A novel microglial subset plays a key role in myelinogenesis in developing brain

Microglia are resident macrophages of the central nervous system that contribute to homeostasis and neuroinflammation. Although known to play an important role in brain development, their exact function has not been fully described. Here, we show that in contrast to healthy adult and inflammation-activated cells, neonatal microglia show a unique myelinogenic and neurogenic phenotype. A CD11c(+) microglial subset that predominates in primary myelinating areas of the developing brain expresses genes for neuronal and glial survival, migration, and differentiation. These cells are the major source of insulin-like growth factor 1, and its selective depletion from CD11c(+) microglia leads to impairment of primary myelination. CD11c-targeted toxin regimens induced a selective transcriptional response in neonates, distinct from adult microglia. CD11c(+) microglia are also found in clusters of repopulating microglia after experimental ablation and in neuroinflammation in adult mice, but despite some similarities, they do not recapitulate neonatal microglial characteristics. We therefore identify a unique phenotype of neonatal microglia that deliver signals necessary for myelination and neurogenesis

Generation of a NG2-EYFP mouse for studying the properties of NG2-expressing cells

A range of vectors were made in which the EYFP gene or the Cre gene were inserted in the start codon of the NG2 gene. The NG2-EYFP vectors were used to generate NG2-EYFP “knockin” mice by homologous recombination. The F1 generation showed lack of EYFP expression, due to NeoR cassette interference. Excision of the NeoR, by breeding the F1 generation to ELLA-Cre mice allowed proper expression of EYFP. NG2-EYFP heterozygous mice were characterized in detail for astrocytic, neurogenic and oligodendrocytic properties through antibody labeling. NG2-EYFP+ cells did not label for the astrocyte marker GFAP, but some cells did express S100 Beta. The cells did not label with any neuronal markers like Beta III tubulin, Neun, and double cortin, but many of the NG2-EYFP+ cells made intimate contacts to the neurons. These contacts are widespread throughout the grey and white matter of the brain. The NG2-EYFP+ cells did label for oligodendrocyte markers like PDGFα-R, NG2, Olig2, O4, and Sox 10. There were a few cells termed phantom cells that did label for NG2, but had no EYFP expression. This could have been caused by improper excision of the NeoR cassette in the F2 generation. The heterozygous mouse is a tool to allow the characterization of the in vivo properties of the NG2+ cells. Breeding of these mice to homozygosity yielded an NG2- knockout mouse, which was also subjected to initial characterization. The NG2-EYFP homozygous showed equivalent cell labeling results to the NG2-EYFP heterozygous mouse, but the phantom cells disappeared in the knockout. The results show that the NG2 cells are a heterogenous population that does not express astrocytic or neuronal markers. The homozygous mouse is an ideal tool to further dissect the properties of the cells, lacking NG2 in vivo

Neurofibromatosis type 2 tumor suppressor protein is expressed in oligodendrocytes and regulates cell proliferation and process formation.

The neurofibromatosis type 2 (NF2) tumor suppressor protein Merlin functions as a negative regulator of cell growth and actin dynamics in different cell types amongst which Schwann cells have been extensively studied. In contrast, the presence and the role of Merlin in oligodendrocytes, the myelin forming cells within the CNS, have not been elucidated. In this work, we demonstrate that Merlin immunoreactivity was broadly distributed in the white matter throughout the central nervous system. Following Merlin expression during development in the cerebellum, Merlin could be detected in the cerebellar white matter tract at early postnatal stages as shown by its co-localization with Olig2-positive cells as well as in adult brain sections where it was aligned with myelin basic protein containing fibers. This suggests that Merlin is expressed in immature and mature oligodendrocytes. Expression levels of Merlin were low in oligodendrocytes as compared to astrocytes and neurons throughout development. Expression of Merlin in oligodendroglia was further supported by its identification in either immortalized cell lines of oligodendroglial origin or in primary oligodendrocyte cultures. In these cultures, the two main splice variants of Nf2 could be detected. Merlin was localized in clusters within the nuclei and in the cytoplasm. Overexpressing Merlin in oligodendrocyte cell lines strengthened reduced impedance in XCELLigence measurements and Ki67 stainings in cultures over time. In addition, the initiation and elongation of cellular projections were reduced by Merlin overexpression. Consistently, cell migration was retarded in scratch assays done on Nf2-transfected oligodendrocyte cell lines. These data suggest that Merlin actively modulates process outgrowth and migration in oligodendrocytes

Merlin affects cell proliferation and migration in a scratch assay.

<p>Scratched areas are covered by new cells in cultures of RT4 (A, D, G), OLN93 (B, E, H) and TC620 (C, F, I) cell lines, stably transfected with pcNF2hflag (<i>Nf2</i>) and pcDNA3.1 (control). (A-C) Images show scratched areas in the culture at the time of scratching and 24 hrs. later. (D-F) To visualize cell morphology in scratched areas after 24 hrs. in culture, higher magnifications are shown. (G-I) Plots shown represent the percentage of the original cell-free area covered by cells after every 4 hours, during a 24 hour period (mean ± SEM, n = 4, *p < 0.05; **p < 0.01, Two-way ANOVA). (J) Cell tracking revealed a significant reduction in total path length between <i>Nf2</i>- and <i>control-</i> transfected cells (mean ± SEM, n_TC620 > 150, n_OLN93 > 200, n_RT4 > 70, **p < 0.01, ***p < 0.001; Wilcoxon rank test). Please note that RT4 cells have run for only 10 hours due to their higher velocity, while OLN93 and TC620 have run for 30 hours. (K) The mitotic index is given as the number of phosphorylated histone H3 cells as compared to total number of cells. No significant difference could be observed between <i>control-</i> and <i>Nf2</i>-transfected cells. Scale bars: (A) 100 μm, (B) 25 μm.</p

Quantification of Merlin expression in cerebellar tissue, cell lines and primary cell cultures.

<p>(A) Merlin expression is demonstrated by Western blotting loading 30 μg of total protein extract. Merlin was expressed as a 70kDa sized protein in RT4, OLN93, TC620 and Oli-neu cell lines; it was absent in SC4 cells. Western blots have been repeated three times with different samples and densitometrically evaluated (B). Expression levels have been related to the expression of Merlin in RT4 cells marked by the dashed line. Significant differences in expression of Merlin in oligodendrocyte cell lines vs. RT4 are marked by asterisks (mean ± SEM, n = 3; *** p < 0.001, ** p < 0.05 t-test).</p

Merlin expression affects cell impedance in XCELLigence measurements.

<p>Cellular impedance was measured in RT4 (A, B), OLN93 (C, D) and TC620 cells (E, F), over-expressing <i>Nf2</i> and in control cells by XCELLigence. The cell index given as a measure of impedance of adhered cells, demonstrated a similar curve in all cell lines (A, C, E). Comparing cell indices over time by evaluating slopes of curves revealed a significant drop in <i>Nf2</i>-transfected (solid line) as compared to the <i>control-</i>transfected cells (dashed line) (B, D, F). Same symbols stand for replicates of one experiment. Lines connect corresponding average values of replicates within a single experiment. Significance values were calculated using the Wilcoxon signed rank test for independent samples (*p < 0.05; **p < 0.01; ***p < 0.001). Pictures in G represent typical images of Ki67 stainings (red). Mitotic figures are depicted by arrows. Total numbers of cells were visualized by Hoechst staining (blue, bar 10 μm). The relative Ki67/Hoechst staining was significantly decreased in <i>Nf2-</i>transfected versus <i>control</i>-transfected cells at 48 and more hours (H), but it was unchanged at shorter time periods (I, ns non-significant).</p

Expression of Merlin and phospho-Merlin in oligodendrocyte cell lines.

<p>RT4, OLN93 and TC620 cell lines were stably transfected with pcNF2hflag (Merlin) and pcDNA3.1 (control). Representative Western blots of gels loaded with 15 μg of total protein extracts are shown in A. The densitometric determination of Merlin and phospho-Merlin in the cell lines was performed by normalizing with cyclophilin B (mean ± SEM, n = 3, ***p < 0.001, Wilcoxon rank-sum test). Immunofluorescent staining of Merlin and phalloidin staining of actin in <i>Nf2</i> stably transfected cell lines demonstrates the preferred localization of Merlin in the cytoplasm (B). Single confocal planes of transfected cells are shown using the same microscope parameters. Scale bars: 10μm.</p