The glucocorticoid dexamethasone inhibits HIF-1α stabilization and metabolic reprogramming in lipopolysaccharide-stimulated primary macrophages

Synthetic glucocorticoids are used to treat many chronic and acute inflammatory conditions. Frequent adverse effects of prolonged exposure toglucocorticoids include disturbances of glucose homeostasis caused by changes in glucose traffic and metabolism in muscle, liver, and adiposetissues. Macrophages are important targets for the anti-inflammatory actions of glucocorticoids. These cells rely on aerobic glycolysis to supportvarious pro-inflammatory and antimicrobial functions. Employing a potent pro-inflammatory stimulus in two commonly used model systems(mouse bone marrow-derived and human monocyte-derived macrophages), we showed that the synthetic glucocorticoid dexamethasone inhib-ited lipopolysaccharide-mediated activation of the hypoxia-inducible transcription factor HIF-1α, a critical driver of glycolysis. In both cell types,dexamethasone-mediated inhibition of HIF-1α reduced the expression of the glucose transporter GLUT1, which imports glucose to fuel aerobicglycolysis. Aside from this conserved response, other metabolic effects of lipopolysaccharide and dexamethasone differed between human andmouse macrophages. These findings suggest that glucocorticoids exert anti-inflammatory effects by impairing HIF-1α-dependent glucose uptakein activated macrophages. Furthermore, harmful and beneficial (anti-inflammatory) effects of glucocorticoids may have a shared mechanisticbasis, depending on the alteration of glucose utilization

DNAAF1 links heart laterality with the AAA+ ATPase RUVBL1 and ciliary intraflagellar transport

DNAAF1 (LRRC50) is a cytoplasmic protein required for dynein heavy chain assembly and cilia motility, and DNAAF1 mutations cause primary ciliary dyskinesia (PCD; MIM 613193). We describe four families with DNAAF1 mutations and complex congenital heart disease (CHD). In three families, all affected individuals have typical PCD phenotypes. However, an additional family demonstrates isolated CHD (heterotaxy) in two affected siblings, but no clinical evidence of PCD. We identified a homozygous DNAAF1 missense mutation, p.Leu191Phe, as causative for heterotaxy in this family. Genetic complementation in dnaaf1-null zebrafish embryos demonstrated the rescue of normal heart looping with wild-type human DNAAF1, but not the p.Leu191Phe variant, supporting the conserved pathogenicity of this DNAAF1 missense mutation. This observation points to a phenotypic continuum between CHD and PCD, providing new insights into the pathogenesis of isolated CHD. In further investigations of the function of DNAAF1 in dynein arm assembly, we identified interactions with members of a putative dynein arm assembly complex. These include the ciliary intraflagellar transport protein IFT88 and the AAA+ (ATPases Associated with various cellular Activities) family proteins RUVBL1 (Pontin) and RUVBL2 (Reptin). Co-localization studies support these findings, with the loss of RUVBL1 perturbing the co-localization of DNAAF1 with IFT88. We show that RUVBL1 orthologues have an asymmetric left-sided distribution at both the mouse embryonic node and the Kupffer’s vesicle in zebrafish embryos, with the latter asymmetry dependent on DNAAF1. These results suggest that DNAAF1-RUVBL1 biochemical and genetic interactions have a novel functional role in symmetry breaking and cardiac development

Generation of a B Cell Model to Investigate the Function of FcRL4 in the Context of RA

Background: FcRL4 is an inhibitory receptor primarily expressed on memory B cells located in mucosal associated lymphoid tissue and in the case of RA, synovial fluid (SF). FcRL4 defines a RANKL-producing B cell, can act as a low affinity IgA receptor and captures IgA IC in inflamed tissue. The objectives of my thesis are to investigate consequences of IgA binding on signal transduction and antigen presentation. To investigate this, a B cell model is useful as cell numbers in SF are relatively low and samples are finite. Inducing continuous expression of FcRL4 in B cell lines leads to growth inhibition so I have designed a model using the Tet-On system, that induces expression of FcRL4 upon doxycycline treatment.Methods: The FcRL4 gene was cloned and inserted into the Tet-On lentiviral vector to generate a doxycycline-inducible vector. The Priess B cell line was transfected and selected using puromycin. FcRL4 expression was induced with doxycycline, cultured overnight and FcRL4+ cells were isolated. FcRL4+ cells were cultured overnight with a citrullinated enolase peptide and HLA-DR4+ T cell hybridoma. Supernatants were collected and IL-2 production measured as a readout for antigen presentation. Results: Transfected Priess cells were able to stably express FcRL4 and its expression had no effect on expression of other co-stimulatory molecules including CD80, CD86 and HLA-DR. Co-culture with the citrullinated enolase peptide resulted in similar levels of IL-2 production by T cells compared to FcRL4- Priess. We are now investigating whether FcRL4 &amp; IgA IC can be internalised using pHrodo avidin and detected in internal compartments. Conclusion: The TetOn lentiviral vector system is suitable for expression of FcRL4 and investigating antigen presentation of a citrullinated peptide. <br/