The microtubule regulator stathmin is an endogenous protein agonist for TLR3.

TLR3 recognizes dsRNAs and is considered of key importance to antiviral host-defense responses. TLR3 also triggers neuroprotective responses in astrocytes and controls the growth of axons and neuronal progenitor cells, suggesting additional roles for TLR3-mediated signaling in the CNS. This prompted us to search for alternative, CNS-borne protein agonists for TLR3. A genome-scale functional screening of a transcript library from brain tumors revealed that the microtubule regulator stathmin is an activator of TLR3-dependent signaling in astrocytes, inducing the same set of neuroprotective factors as the known TLR3 agonist polyinosinic:polycytidylic acid. This activity of stathmin crucially depends on a long, negatively charged alpha helix in the protein. Colocalization of stathmin with TLR3 on astrocytes, microglia, and neurons in multiple sclerosis-affected human brain indicates that as an endogenous TLR3 agonist, stathmin may fulfill previously unsuspected regulatory roles during inflammation and repair in the adult CNS.

Development of atopic dermatitis in mice transgenic for human apolipoprotein C1.

Mice with transgenic expression of human apolipoprotein C1 (APOC1) in liver and skin have strongly increased serum levels of cholesterol, triglycerides, and free fatty acids, indicative of a disturbed lipid metabolism. Importantly, these mice display a disturbed skin barrier function, evident from increased transepidermal water loss, and spontaneously develop symptoms of dermatitis including scaling, lichenification, excoriations, and pruritus. Histological analysis shows increased epidermal thickening and spongiosis in conjunction with elevated numbers of inflammatory cells (eosinophils, neutrophils, mast cells, macrophages, and CD4+ T cells) in the dermis. In addition, affected mice have increased serum levels of IgE and show abundant IgE(+) mast cells in the dermis. Partial inhibition of disease could be achieved by restoration of the skin barrier function with topical application of a lipophilic ointment. Furthermore, the development of atopic dermatitis in these mice was suppressed by corticosteroid treatment. These findings in APOC1(+/+) mice underscore the role of skin barrier integrity in the pathogenesis of atopic dermatitis.

Differentiation of primary adult microglia alters their response to TLR8-mediated activation but not their capacity as APC.

Activated microglia are found in a variety of neuroinflammatory disorders where they have attributed roles as effector as well as antigen-presenting cells (APC). Critical determinants for the multifaceted role of microglia are the differentiation potential of microglia and their mode of activation. In this study, we have investigated the effects of M-CSF and GM-CSF-mediated differentiation of adult primate microglia on their cellular phenotype, antigen presentation, and phagocytic function as well as on Toll-like receptor (TLR)-mediated responses. We show that although cell morphology and expression levels of activation markers were markedly different, differentiation with either factor yielded microglia that phenotypically and functionally resemble macrophages. Both M-CSF and GM-CSF-differentiated microglia were responsive to TLR1/2, 2, 3, 4, 5, 6/2, and 8-mediated activation, but not to TLR7 or 9-mediated activation. Intriguingly, M-CSF-differentiated microglia expressed higher levels of TLR8-encoding mRNA and protein, and produced larger amounts of proinflammatory cytokines in response to TLR8-mediated activation as compared to GM-CSF-differentiated microglia. While differentiation of adult microglia by growth factors that can be produced endogenously in the central nervous system is thus unlikely to change their APC function, it can alter their innate responses to infectious stimuli such as ssRNA viruses. Resident primate microglia may thereby help shape rather than initiate adaptive immune responses.

Toll-like receptor 3 on adult human astrocytes triggers production of neuroprotective mediators.

Toll-like receptors (TLRs) are innate immunity receptors that are expressed on a wide range of cell types, including CNS glial cells. In general, TLR engagement by specific sets of microbial ligands triggers production of pro-inflammatory factors and enhances antigen-presenting cell functions. The functional roles of TLR in the CNS, however, are still poorly understood. While adult human astrocytes in culture dominantly express TLR4, they display a strikingly strong and selective induction of TLR3 when activated by pro-inflammatory cytokines, TLR3 or TLR4 agonists, or oxidative stress. Gene profiling analysis of the astrocyte response to either TLR3 or TLR4 activation revealed that TLR3, but not TLR4, induces expression of a range of neuroprotective mediators and several other molecules that regulate cellular growth, differentiation, and migration. Also, TLR3 triggered enhanced production of anti-inflammatory cytokines including interleukin-9 (IL-9), IL-10, and IL-11 and downregulation of the p40 subunit of IL-12 and IL-23. The collective TLR3-induced products were found in functional assays to inhibit astrocyte growth, promote human endothelial cell growth, and importantly, to enhance neuronal survival in organotypic human brain slice cultures. Together, our data indicate that TLR3 is induced on human astrocytes upon inflammation and when activated, mediates a comprehensive neuroprotective response rather than a polarized pro-inflammatory reaction.

Cultured human adult microglia from different donors display stable cytokine, chemokine and growth factor gene profiles but respond differently to a pro-inflammatory stimulus.

OBJECTIVES: Brain microglia are highly responsive cells in the central nervous system that exert key functions in host defense as well as in neuroprotection and regeneration. In this study the gene expression profiles for 268 cytokines, chemokines, growth factors and their receptors were examined in cultures of purified human adult microglia, using cDNA array profiling. METHODS: Microglia from 9 different donors were compared, also following challenge of such microglia with the pro-inflammatory cytokines TNF-alpha and IFN-gamma. RESULTS: A stable pattern was observed of genes abundantly expressed in the different cultures under standard conditions. Genes abundantly expressed in all microglia cultures include CCL2 (MCP-1), thymosin beta-10, migration-inhibitory factor-related protein 8 (MRP8), MRP14, corticotropin-releasing factor receptor 1 and endothelin 2. Abundant gene products novel to microglia were neuromodulin (GAP43) and Flt3 ligand. Yet, treatment with TNF-alpha and IFN-gamma led to widely different response profiles among the different cultures. CONCLUSION: These data show a surprising level of heterogeneity among human adult microglia cultures in their response to a pro-inflammatory stimulus despite the standardized methodology to examine this response.

Modulation of the cytokine network in human adult astrocytes by human herpesvirus-6A.

Human herpesvirus-6A (HHV-6A) is a common pathogen whose role in CNS disorders including multiple sclerosis remains controversial. To understand how HHV-6A could influence inflammatory pathways in the CNS, we infected cultured human adult astrocytes and examined the expression of 268 cytokines, chemokines, growth factors and their receptors by gene profiling. HHV-6 infection alone had little effect on the astrocyte gene profile but strongly altered the astrocyte response to proinflammatory cytokines. Under those conditions astrocytes express higher levels of anti-inflammatory mediators including IL-10 and IL-11, chemotactic factors, growth factors and factors controlling type I interferon production. Our data suggest that HHV-6 itself does not evoke a pro-inflammatory response in astrocytes but rather triggers immune modulatory factors in the face of inflammation.

Cytokine, chemokine and growth factor gene profiling of cultured human astrocytes after exposure to proinflammatory stimuli.

Astrocytes play key roles in CNS development, inflammation, and repair by producing a wide variety of cytokines, chemokines, and growth factors. Understanding the regulation of this network is important for a full understanding of astrocyte functioning. In this study, expression levels of 268 genes encoding cytokines, chemokines, growth factors, and their receptors were established in cultured human adult astrocytes using cDNA arrays. Also, changes in this gene profile were determined following stimulation with TNFalpha, IL-1beta, and IFNgamma. The data obtained reveal a highly reproducible pattern of gene expression not only between different astrocyte cultures from a single source, but also between astrocytes from different donors. They also identify several gene products not previously described for human astrocytes, including a.o. IL-17, CD70, CD147, and BIGH3. When stimulated with TNFalpha astrocytes respond with increased expression of several genes, notably including those encoding the chemokines CCL2 (MCP-1), CCL5 (RANTES), and CXCL8 (IL-8), growth factors including BMP-2A, BMP-3, neuromodulin (GAP43), BDNF, and G-CSF, and receptors such as the CRF receptor, the calcitonin receptor (CTR), and TKT. The response to IL-1beta involves largely the same range of genes, but responses were blunted in comparison to the TNFalpha response. Treatment with IFNgamma had no or only marginal effects on expression of any of the 268 genes analyzed. Astrocytes treated with a mixture of all three stimuli together displayed responses that are largely similar to those found in response to TNFalpha or IL-1beta alone, with only few additional synergistic effects.