N-Wasp Regulates Oligodendrocyte Myelination.

Oligodendrocyte myelination depends on actin cytoskeleton rearrangement. Neural Wiskott-Aldrich syndrome protein(N-Wasp) is an actin nucleation factor that promotes polymerization of branched actin filaments. N-Wasp activity is essential for myelin membrane wrapping by Schwann cells, but its role in oligodendrocytes and CNS myelination remains unknown. Here we report that oligodendrocytes-specific deletion of N-Wasp in mice of both sexes resulted in hypomyelination (i.e., reduced number of myelinated axons and thinner myelin profiles), as well as substantial focal hypermyelination reflected by the formation of remarkably long myelin outfolds. These myelin outfolds surrounded unmyelinated axons, neuronal cell bodies, and other myelin profiles. The latter configuration resulted in pseudo-multimyelin profiles that were often associated with axonal detachment and degeneration throughout the CNS, including in the optic nerve, corpus callosum, and the spinal cord. Furthermore, developmental analysis revealed that myelin abnormalities were already observed during the onset of myelination, suggesting that they are formed by aberrant and misguided elongation of the oligodendrocyte inner lip membrane. Our results demonstrate that N-Wasp is required for the formation of normal myelin in the CNS. They also reveal that N-Wasp plays a distinct role in oligodendrocytes compared with Schwann cells, highlighting a difference in the regulation of actin dynamics during CNS and PNS myelination.SIGNIFICANCE STATEMENT Myelin is critical for the normal function of the nervous system by facilitating fast conduction of action potentials. During the process of myelination in the CNS, oligodendrocytes undergo extensive morphological changes that involve cellular process extension and retraction, axonal ensheathment, and myelin membrane wrapping. Here we present evidence that N-Wasp, a protein regulating actin filament assembly through Arp2/3 complex-dependent actin nucleation, plays a critical role in CNS myelination, and its absence leads to several myelin abnormalities. Our data provide an important step into the understanding of the molecular mechanisms underlying CNS myelination.

Regulation of the inflammatory profile of stromal cells in human breast cancer: prominent roles for TNF-α and the NF-κB pathway.

INTRODUCTION: Breast cancer progression is promoted by stromal cells that populate the tumors, including cancer-associated fibroblasts (CAFs) and mesenchymal stem/stromal cells (MSCs). The activities of CAFs and MSCs in breast cancer are integrated within an intimate inflammatory tumor microenvironment (TME) that includes high levels of tumor necrosis factor α (TNF-α) and interleukin 1ß (IL-1ß). Here, we identified the impact of TNF-α and IL-1ß on the inflammatory phenotype of CAFs and MSCs by determining the expression of inflammatory chemokines that are well-characterized as pro-tumorigenic in breast cancer: CCL2 (MCP-1), CXCL8 (IL-8) and CCL5 (RANTES). METHODS: Chemokine expression was determined in breast cancer patient-derived CAFs by ELISA and in patient biopsies by immunohistochemistry. Chemokine levels were determined by ELISA in (1) human bone marrow-derived MSCs stimulated by tumor conditioned media (Tumor CM) of breast tumor cells (MDA-MB-231 and MCF-7) at the end of MSC-to-CAF-conversion process; (2) Tumor CM-derived CAFs, patient CAFs and MSCs stimulated by TNF-α (and IL-1ß). The roles of AP-1 and NF-κB in chemokine secretion were analyzed by Western blotting and by siRNAs to c-Jun and p65, respectively. Migration of monocytic cells was determined in modified Boyden chambers. RESULTS: TNF-α (and IL-1ß) induced the release of CCL2, CXCL8 and CCL5 by MSCs and CAFs generated by prolonged stimulation of MSCs with Tumor CM of MDA-MB-231 and MCF-7 cells. Patient-derived CAFs expressed CCL2 and CXCL8, and secreted CCL5 following TNF-α (and IL-1ß) stimulation. CCL2 was expressed in CAFs residing in proximity to breast tumor cells in biopsies of patients diagnosed with invasive ductal carcinoma. CCL2 release by TNF-α-stimulated MSCs was mediated by TNF-RI and TNF-RII, through the NF-κB but not via the AP-1 pathway. Exposure of MSCs to TNF-α led to potent CCL2-induced migration of monocytic cells, a process that may yield pro-cancerous myeloid infiltrates in breast tumors. CONCLUSIONS: Our novel results emphasize the important roles of inflammation-stroma interactions in breast cancer, and suggest that NF-κB may be a potential target for inhibition in tumor-adjacent stromal cells, enabling improved tumor control in inflammation-driven malignancies.

Reduced expression of chemoattractant receptors by polymorphonuclear leukocytes in Hyper IgE Syndrome patients.

Hyper IgE Syndrome (HIES) is a rare genetic disorder, characterized by elevated serum IgE levels and reduced inflammatory responses to bacterial infections. This leads to dermatitis, recurrent lung infections and "cold abscesses". Recently, progress was made in HIES research, when mutations in STAT3 were found in the autosomal dominant form of HIES, and impaired responses of T helper 17 cells were reported. However, the causes for reduced inflammatory responses in these patients were not fully elucidated. In view of studies that indicated that polymorphonuclear leukocytes (PMN) of HIES patients are defective in their chemotactic properties, we asked if the PMN of these patients have reduced expression of receptors for chemoattractants. To analyze this possibility, we focused on fMLP and ELR(+)-CXC chemokines - which are essential for mounting acute inflammatory responses - and determined the coding sequences and expression levels of their corresponding receptors: FPR (for fMLP) as well as CXCR1 and CXCR2 (the receptors for ELR(+)-CXC chemokines). The analyses of these receptors in HIES patients indicated that their coding sequences were intact and normal. However, the percentages of PMN that expressed FPR, CXCR1 and CXCR2 were significantly lower in HIES patients. In addition, lower expression levels per cell were denoted for CXCR1 in PMN of the patients. A cumulative score that was calculated for the three chemoattractant receptors together indicated that in some of the patients there were prominent reductions, of up to approximately 50% in the overall expression of the receptors (indicated by % positive cells and mean expression levels per cell). In addition, we asked whether deregulation of PMN activities in HIES may result from binding of IgE to corresponding receptors on HIES PMN. Our findings indicate that this is probably not the case, because similarly to normal PMN, the cells of HIES patients did not express notable levels of the IgE receptors FcvarepsilonRI and FcvarepsilonRII. Together, these results provide novel information on the expression of key determinants in PMN migration in HIES, suggesting that a defect in the expression of chemoattractant receptors may lead to impaired chemotaxis found in HIES patients, and to decreased inflammatory responses.