The ketone body b-hydroxybutyrate (BHB) is an endogenous factor protecting against stroke and neurodegenerative diseases, but its mode of action is unclear. Here we show in a stroke model that the hydroxy-carboxylic acid receptor 2 (HCA 2 , GPR109A) is required for the neuroprotective effect of BHB and a ketogenic diet, as this effect is lost in Hca2 À / À mice. We further demonstrate that nicotinic acid, a clinically used HCA 2 agonist, reduces infarct size via a HCA 2 -mediated mechanism, and that noninflammatory Ly-6C Lo monocytes and/or macrophages infiltrating the ischemic brain also express HCA 2 . Using cell ablation and chimeric mice, we demonstrate that HCA 2 on monocytes and/or macrophages is required for the protective effect of nicotinic acid. The activation of HCA 2 induces a neuroprotective phenotype of monocytes and/or macrophages that depends on PGD 2 production by COX1 and the haematopoietic PGD 2 synthase. Our data suggest that HCA 2 activation by dietary or pharmacological means instructs Ly-6C Lo monocytes and/or macrophages to deliver a neuroprotective signal to the brain.

Fibrillin-1 and fibrillin-2 constitute the backbone of extracellular filaments, called microfibrils. Fibrillin assembly involves complex multistep mechanisms to result in a periodical head-to-tail alignment in microfibrils. Impaired assembly potentially plays a role in the molecular pathogenesis of genetic disorders caused by mutations in fibrillin-1 (Marfan syndrome) and fibrillin-2 (congenital contractural arachnodactyly). Presently, the basic molecular interactions involved in fibrillin assembly are obscure. Here, we have generated recombinant full-length human fibrillin-1, and two overlapping recombinant polypeptides spanning the entire human fibrillin-2 in a mammalian expression system. Characterization by gel electrophoresis, electron microscopy after rotary shadowing, and reactivity with antibodies demonstrated correct folding of these recombinant polypeptides. Analyses of homotypic and heterotypic interaction repertoires showed N-to C-terminal binding of fibrillin-1, and of fibrillin-1 with fibrillin-2. The interactions were of high affinity with dissociation constants in the low nanomolar range. However, the Nand C-terminal fibrillin-2 polypeptides did not interact with each other. These results demonstrate that fibrillins can directly interact in an N-to C-terminal fashion to form homotypic fibrillin-1 or heterotypic fibrillin-1/ fibrillin-2 microfibrils. This conclusion was further strengthened by double immunofluorescence labeling of microfibrils. In addition, the binding epitopes as well as the entire fibrillin molecules displayed very stable properties.

Many of the biological properties of C5a are mediated through activation of its receptor (C5aR1), the expression of which has been demonstrated convincingly on myeloid cells, such as neutrophils, monocytes, and macrophages. In contrast, conflicting results exist regarding C5aR1 expression in dendritic cells (DCs) and lymphoid lineage cells. In this article, we report the generation of a floxed GFP-C5aR1 reporter knock-in mouse. Using this mouse strain, we confirmed strong C5aR1 expression in neutrophils from bone marrow, blood, lung, and spleen, as well as in peritoneal macrophages. Further, we show C5aR1 expression in lung eosinophils, lung- and lamina propria–resident and alveolar macrophages, bone marrow–derived DCs, and lung-resident CD11b+ and monocyte-derived DCs, whereas intestinal and pulmonary CD103+ DCs stained negative. Also, some splenic NKT cells expressed GFP, whereas naive NK cells and B2 cells lacked GFP expression. Finally, we did not observe any C5aR1 expression in naive or activated CD4+ Th cells in vitro or in vivo. Mating the floxed GFP-C5aR1 mouse strain with LysMCre mice, we were able to specifically delete C5aR1 in neutrophils and macrophages, whereas C5aR1 expression was retained in DCs. In summary, our findings suggest that C5aR1 expression in mice is largely restricted to cells of the myeloid lineage. The novel floxed C5aR1 reporter knock-in mouse will prove useful to track C5aR1 expression in experimental models of acute and chronic inflammation and to conditionally delete C5aR1 in immune cells.

C3a exerts multiple biologic functions through activation of its cognate C3a receptor. C3 and C3aR mice have been instrumental in defining important roles of the C3a/C3aR axis in the regulation of acute and chronic inflammatory diseases, including ischemia/reperfusion injury, allergic asthma, autoimmune nephritis, and rheumatoid arthritis. Surprisingly little is known about C3aR expression and function in immune and stromal cells. To close this gap, we generated a floxed tandem-dye Tomato (tdTomato)-C3aR reporter knock-in mouse, which we used to monitor C3aR expression in cells residing in the lung, airways, lamina propria (LP) of the small intestine, brain, visceral adipose tissue, bone marrow (BM), spleen, and the circulation. We found a strong expression of tdTomato-C3aR in the brain, lung, LP, and visceral adipose tissue, whereas it was minor in the spleen, blood, BM, and the airways. Most macrophage and eosinophil populations were tdTomato-C3aR Interestingly, most tissue eosinophils and some macrophage populations expressed C3aR intracellularly. BM-derived dendritic cells (DCs), lung-resident cluster of differentiation (CD) 11b conventional DCs (cDCs) and monocyte-derived DCs, LP CD103, and CD11b cDCs but not pulmonary CD103 cDCs and splenic DCs were tdTomato-C3aR Surprisingly, neither BM, blood, lung neutrophils, nor mast cells expressed C3aR. Similarly, all lymphoid-derived cells were tdTomato-C3aR, except some LP-derived type 3 innate lymphoid cells. Pulmonary and LP-derived epithelial cells expressed at best minor levels of C3aR. In summary, we provide novel insights into the expression pattern of C3aR in mice. The floxed C3aR knock-in mouse will help to reliably track and conditionally delete C3aR expression in experimental models of inflammation.

Mutations in fibrillin-1 lead to Marfan syndrome and some related genetic disorders. Many of the more than 600 mutations currently known in fibrillin-1 eliminate or introduce cysteine residues in epidermal growth factor-like modules. Here we report structural and functional consequences of three selected cysteine mutations (R627C, C750G, and C926R) in fibrillin-1. The mutations have been analyzed by means of recombinant polypeptides produced in mammalian expression systems. The mRNA levels for the mutation constructs were similar to wild-type levels. All three mutated polypeptides were secreted by embryonic kidney cells (293) into the culture medium. Purification was readily feasible for mutants R627C and C750G, but not for C926R, which restricted the availability of this mutant polypeptide to selected analyses. The overall folds of the mutant polypeptides were indistinguishable from the wild-type as judged by the ultrastructural shape, CD analysis, and reactivity with a specific antibody sensitive for intact disulfide bonds. Subtle structural changes caused by R627C and C750G, however, were monitored by proteolysis and heat denaturation experiments. These changes occurred in the vicinity of the mutations either as short range effects (R627C) or both short and long range effects (C750G). Enhanced proteolytic susceptibility was observed for R627C and C750G to a variety of proteases. These results expand and further strengthen the concept that proteolytic degradation of mutated fibrillin-1 might be an important potential mechanism in the pathogenesis of Marfan syndrome and other disorders caused by mutations in fibrillin-1.