Cell-specific deletion of C1qa identifies microglia as the dominant source of C1q in mouse brain

BACKGROUND: The complement cascade not only provides protection from infection but can also mediate destructive inflammation. Complement is also involved in elimination of neuronal synapses which is essential for proper development, but can be detrimental during aging and disease. C1q, required for several of these complement-mediated activities, is present in the neuropil, microglia, and a subset of interneurons in the brain. METHODS: To identify the source(s) of C1q in the brain, the C1qa gene was selectively inactivated in the microglia or Thy-1(+) neurons in both wild type mice and a mouse model of Alzheimer’s disease (AD), and C1q synthesis assessed by immunohistochemistry, QPCR, and western blot analysis. RESULTS: While C1q expression in the brain was unaffected after inactivation of C1qa in Thy-1(+) neurons, the brains of C1qa (FL/FL) :Cx3cr1 (CreERT2) mice in which C1qa was ablated in microglia were devoid of C1q with the exception of limited C1q in subsets of interneurons. Surprisingly, this loss of C1q occurred even in the absence of tamoxifen by 1 month of age, demonstrating that Cre activity is tamoxifen-independent in microglia in Cx3cr1 (CreERT2/WganJ) mice. C1q expression in C1qa (FL/FL) : Cx3cr1 (CreERT2/WganJ) mice continued to decline and remained almost completely absent through aging and in AD model mice. No difference in C1q was detected in the liver or kidney from C1qa (FL/FL) : Cx3cr1 (CreERT2/WganJ) mice relative to controls, and C1qa (FL/FL) : Cx3cr1 (CreERT2/WganJ) mice had minimal, if any, reduction in plasma C1q. CONCLUSIONS: Thus, microglia, but not neurons or peripheral sources, are the dominant source of C1q in the brain. While demonstrating that the Cx3cr1 (CreERT2/WganJ) deleter cannot be used for adult-induced deletion of genes in microglia, the model described here enables further investigation of physiological roles of C1q in the brain and identification of therapeutic targets for the selective control of complement-mediated activities contributing to neurodegenerative disorders. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12974-017-0814-9) contains supplementary material, which is available to authorized users

Blocking Synthesis of the Variant Surface Glycoprotein Coat in Trypanosoma brucei Leads to an Increase in Macrophage Phagocytosis Due to Reduced Clearance of Surface Coat Antibodies

The extracellular bloodstream form parasite Trypanosoma brucei is supremely adapted to escape the host innate and adaptive immune system. Evasion is mediated through an antigenically variable Variant Surface Glycoprotein (VSG) coat, which is recycled at extraordinarily high rates. Blocking VSG synthesis triggers a precytokinesis arrest where stalled cells persist for days in vitro with superficially intact VSG coats, but are rapidly cleared within hours in mice. We therefore investigated the role of VSG synthesis in trypanosome phagocytosis by activated mouse macrophages. T. brucei normally effectively evades macrophages, and induction of VSG RNAi resulted in little change in phagocytosis of the arrested cells. Halting VSG synthesis resulted in stalled cells which swam directionally rather than tumbling, with a significant increase in swim velocity. This is possibly a consequence of increased rigidity of the cells due to a restricted surface coat in the absence of VSG synthesis. However if VSG RNAi was induced in the presence of anti-VSG221 antibodies, phagocytosis increased significantly. Blocking VSG synthesis resulted in reduced clearance of anti-VSG antibodies from the trypanosome surface, possibly as a consequence of the changed motility. This was particularly marked in cells in the G2/ M cell cycle stage, where the half-life of anti-VSG antibody increased from 39.3 ± 4.2 seconds to 99.2 ± 15.9 seconds after induction of VSG RNAi. The rates of internalisation of bulk surface VSG, or endocytic markers like transferrin, tomato lectin or dextran were not significantly affected by the VSG synthesis block. Efficient elimination of anti-VSG-antibody complexes from the trypanosome cell surface is therefore essential for trypanosome evasion of macrophages. These experiments highlight the essentiality of high rates of VSG recycling for the rapid removal of host opsonins from the parasite surface, and identify this process as a key parasite virulence factor during a chronic infection

Fundamental role of C1q in autoimmunity and inflammation

C1q, historically viewed as the initiating component of the classical complement pathway, also exhibits a variety of complement-independent activities in both innate and acquired immunity. Recent studies focusing on C1q\u27s suppressive role in the immune system have provided new insight into how abnormal C1q expression and bioactivity may contribute to autoimmunity. In particular, molecular networks involving C1q interactions with cell surface receptors and other ligands are emerging as mechanisms involved in C1q\u27s modulation of immunity. Here, we discuss the role of C1q in controlling immune cell function, including recently elucidated mechanisms of action, and suggest how these processes are critical for maintaining tissue homeostasis under steady-state conditions and in preventing autoimmunity

Identification of Markers that Distinguish Monocyte-Derived Fibrocytes from Monocytes, Macrophages, and Fibroblasts

The processes that drive fibrotic diseases are complex and include an influx of peripheral blood monocytes that can differentiate into fibroblast-like cells called fibrocytes. Monocytes can also differentiate into other cell types, such as tissue macrophages. The ability to discriminate between monocytes, macrophages, fibrocytes, and fibroblasts in fibrotic lesions could be beneficial in identifying therapies that target either stromal fibroblasts or fibrocytes. and in sections from human lung. We found that markers such as CD34, CD68, and collagen do not effectively discriminate between the four cell types. In addition, IL-4, IL-12, IL-13, IFN-γ, and SAP differentially regulate the expression of CD32, CD163, CD172a, and CD206 on both macrophages and fibrocytes. Finally, CD49c (α3 integrin) expression identifies a subset of fibrocytes, and this subset increases with time in culture.These results suggest that discrimination of monocytes, macrophages, fibrocytes, and fibroblasts in fibrotic lesions is possible, and this may allow for an assessment of fibrocytes in fibrotic diseases

Multimerin-2 is a ligand for group14 family C-type lectins CLEC14A, CD93 and CD248 spanning the endothelial pericyte interface:MMRN2 binds three group 14 family C-type lectins

The C-type lectin domain containing group 14 family members CLEC14A and CD93 are proteins expressed by endothelium and are implicated in tumour angiogenesis. CD248 (alternatively known as endosialin or tumour endothelial marker-1) is also a member of this family and is expressed by tumour-associated fibroblasts and pericytes. Multimerin-2 (MMRN2) is a unique endothelial specific extracellular matrix protein that has been implicated in angiogenesis and tumour progression. We show that the group 14 C-type lectins CLEC14A, CD93 and CD248 directly bind to MMRN2 and only thrombomodulin of the family does not. Binding to MMRN2 is dependent on a predicted long-loop region in the C-type lectin domain and is abrogated by mutation within the domain. CLEC14A and CD93 bind to the same non-glycosylated coiled-coil region of MMRN2, but the binding of CD248 occurs on a distinct non-competing region. CLEC14A and CD248 can bind MMRN2 simultaneously and this occurs at the interface between endothelium and pericytes in human pancreatic cancer. A recombinant peptide of MMRN2 spanning the CLEC14A and CD93 binding region blocks CLEC14A extracellular domain binding to the endothelial cell surface as well as increasing adherence of human umbilical vein endothelial cells to the active peptide. This MMRN2 peptide is anti-angiogenic in vitro and reduces tumour growth in mouse models. These findings identify novel protein interactions involving CLEC14A, CD93 and CD248 with MMRN2 as targetable components of vessel formation.</p