Differential requirements of MyD88 and TRIF pathways in TLR4-mediated immune responses in murine B cells

LPS stimulates the TLR4/Myeloid differentiation protein-2 (MD-2) complex and promotes a variety ofimmune responses in B cells. TLR4 has two main signaling pathways, MyD88 and Toll/IL-1R (TIR)-domain-containing adaptor-inducing interferon- (TRIF) pathways, but relatively few studies have examinedthese pathways in B cells. In this study, we investigated MyD88- or TRIF-dependent LPS responses inB cells by utilizing their knockout mice. Compared with wild-type (WT) B cells, MyD88−/−B cells weremarkedly impaired in up-regulation of CD86 and proliferation induced by lipid A moiety of LPS. TRIF−/−Bcells were also impaired in these responses compared with WT B cells, but showed better responses thanMyD88−/−B cells. Regarding class switch recombination (CSR) elicited by lipid A plus IL-4, MyD88−/−B cells showed similar patterns of CSR to WT B cells. However, TRIF−/−B cells showed the impaired inthe CSR. Compared with WT and MyD88−/−B cells, TRIF−/−B cells exhibited reduced cell division, fewerIgG1+cells per division, and decreased activation-induced cytidine deaminase (Aicda) mRNA expressionin response to lipid A plus IL-4. Finally, IgG1 production to trinitrophenyl (TNP)-LPS immunization wasimpaired in TRIF−/−mice, while MyD88−/−mice exhibited increased IgG1 production. Thus, MyD88 andTRIF pathways differently regulate TLR4-induced immune responses in B cells

Construction of 3D models of the CYP11B family as a tool to predict ligand binding characteristics

Aldosterone is synthesised by aldosterone synthase (CYP11B2). CYP11B2 has a highly homologous isoform, steroid 11β-hydroxylase (CYP11B1), which is responsible for the biosynthesis of aldosterone precursors and glucocorticoids. To investigate aldosterone biosynthesis and facilitate the search for selective CYP11B2 inhibitors, we constructed three-dimensional models for CYP11B1 and CYP11B2 for both human and rat. The models were constructed based on the crystal structure of Pseudomonas Putida CYP101 and Oryctolagus Cuniculus CYP2C5. Small steric active site differences between the isoforms were found to be the most important determinants for the regioselective steroid synthesis. A possible explanation for these steric differences for the selective synthesis of aldosterone by CYP11B2 is presented. The activities of the known CYP11B inhibitors metyrapone, R-etomidate, R-fadrazole and S-fadrazole were determined using assays of V79MZ cells that express human CYP11B1 and CYP11B2, respectively. By investigating the inhibitors in the human CYP11B models using molecular docking and molecular dynamics simulations we were able to predict a similar trend in potency for the inhibitors as found in the in vitro assays. Importantly, based on the docking and dynamics simulations it is possible to understand the enantioselectivity of the human enzymes for the inhibitor fadrazole, the R-enantiomer being selective for CYP11B2 and the S-enantiomer being selective for CYP11B1