Commensal Bacteria-induced Interleukin 1β (IL-1β) Secreted by Macrophages Up-regulates Hepcidin Expression in Hepatocytes by Activating the Bone Morphogenetic Protein Signaling Pathway

The liver hormone hepcidin is the central regulator of systemic iron metabolism. Its increased expression in inflammatory states leads to hypoferremia and anemia. Elucidation of the mechanisms that up-regulate hepcidin during inflammation is essential for developing rational therapies for this anemia. Using mouse models of inflammatory bowel disease, we have shown previously that colitis-associated hepcidin induction is influenced by intestinal microbiota composition. Here we investigate how two commensal bacteria, Bifidobacterium longum and Bacteroides fragilis, representative members of the gut microbiota, affect hepcidin expression. We found that supernatants of a human macrophage cell line infected with either of the bacteria up-regulated hepcidin when added to a human hepatocyte cell line. This activity was abrogated by neutralization of IL-1β. Moreover, purified IL-1β increased hepcidin expression when added to the hepatocyte line or primary human hepatocytes and when injected into mice. IL-1β activated the bone morphogenetic protein (BMP) signaling pathway in hepatocytes and in mouse liver, as indicated by increased phosphorylation of small mothers against decapentaplegic proteins. Activation of BMP signaling correlated with IL-1β-induced expression of BMP2 in human hepatocytes and activin B in mouse liver. Treatment of hepatocytes with two different chemical inhibitors of BMP signaling or with a neutralizing antibody to BMP2 prevented IL-1β-induced up-regulation of hepcidin. Our results clarify how commensal bacteria affect hepcidin expression and reveal a novel connection between IL-1β and activation of BMP signaling. They also suggest that there may be differences between mice and humans with respect to the mechanism by which IL-1β up-regulates hepcidin.     

The L33F darunavir resistance mutation acts as a molecular anchor reducing the flexibility of the HIV-1 protease 30s and 80s loops

HIV-1 protease (PR) is a 99 amino acid protein responsible for proteolytic processing of the viral polyprotein – an essential step in the HIV-1 life cycle. Drug resistance mutations in PR that are selected during antiretroviral therapy lead to reduced efficacy of protease inhibitors (PI) including darunavir (DRV). To identify the structural mechanisms associated with the DRV resistance mutation L33F, we performed X-ray crystallographic studies with a multi-drug resistant HIV-1 protease isolate that contains the L33F mutation (MDR769 L33F). In contrast to other PR L33F DRV complexes, the structure of MDR769 L33F complexed with DRV reported here displays the protease flaps in an open conformation. The L33F mutation increases noncovalent interactions in the hydrophobic pocket of the PR compared to the wild-type (WT) structure. As a result, L33F appears to act as a molecular anchor, reducing the flexibility of the 30s loop (residues 29–35) and the 80s loop (residues 79–84). Molecular anchoring of the 30s and 80s loops leaves an open S1/S1′ subsite and distorts the conserved hydrogen-bonding network of DRV. These findings are consistent with previous reports despite structural differences with regards to flap conformation.     

IL-38: A new factor in rheumatoid arthritis

The newly characterized cytokine IL-38 (IL-1F10) belongs to the IL-1 family of cytokines. Previous work has demonstrated that IL-38 inhibited Candida albicans-induced IL-17 production from peripheral blood mononuclear cells. However, it is still unclear whether IL-38 is an inflammatory or an anti-inflammatory cytokine. We generated anti-human IL-38 monoclonal antibodies in order to perform immunohistochemical staining and an enzyme-linked immunosorbent assay. While human recombinant IL-38 protein was not cleaved by recombinant caspase-1, chymase, or PR3 in vitro, overexpression of IL-38 cDNA produced a soluble form of IL-38 protein. Furthermore, immunohistochemical analysis showed that synovial tissues obtained from RA patients strongly expressed IL-38 protein. To investigate the biological role of IL-38, C57BL/6 IL-38 gene-deficient (^?/?) mice were used in an autoantibody-induced rheumatoid arthritis (RA) mouse model. As compared with control mice, IL-38 ^(?/?) mice showed greater disease severity, accompanied by higher IL-1β and IL-6 gene expression in the joints. Therefore, IL-38 acts as an inhibitor of the pathogenesis of autoantibody-induced arthritis in mice and may have a role in the development or progression of RA in humans.     

InSAC: A novel sub-nuclear body essential for Interleukin-6 and -10 RNA processing and stability

Dysregulation of cytokine expression causes inflammatory diseases or chronic infection conditions. We have identified that Tat-activating regulatory DNA-binding protein-43 (TDP-43) is involved in cytokine RNA processing in order to promote an optimal immune response. The interaction of TDP-43 with spliceosomal components from the Cajal body leads to the formation of a novel sub-nuclear body called the Interleukin (IL)-6 and IL-10 Splicing Activating Compartment (InSAC). TDP-43 binds to the IL-6 and IL-10 RNAs in a sequence-dependent manner. In cell-based studies, we observed that lipopoly-saccharide (LPS) stimulation induces the formation of the InSAC through TDP-43 ubiquitination, thereby influencing the processing and expression levels of IL-6 RNA. Moreover, TDP-43 knockdown in vivo results in a decrease in IL-6 production and its RNA splicing and stability. Thus, these findings demonstrate that the InSAC is linked to the activation and modulation of the immune response. [BMB Reports 2015; 48(5): 239-240]     

Selective separation procedure for determination of ribosomal proteins L7 and L12.

An electrophoretic procedure for the selective separation and determination of the closely similar ribosomal proteins L7 and L12 (which are specifically involved in the GTPase reactions of the ribosome) from the total protein mixture extracted from unwashed ribosomes is described. In this procedure, which takes advantage of their unusually low isoelectric points. L7 and L12 (and a few other acidic proteins) migrate into gel asanions, while the bulk of ribosomal proteins which are basic remain behind. The positions of L7 and L12 were determined with authentic, pure proteins. It was further determined by means of 2-dimensional gel electrophoresis that no other protein components present in unwashed ribosomes comigrate with the bands of L7 and L12.     

Continuous infusion of cholecystokinin and meal pattern in the rat

Purified natural cholecystokinin (CCK-33) was infused continuously for two days at a rate of 5.9 μg/hr in two rats trained to bar-press for food (Noyes pellet 45 mg) on a fixed ratio of five bar presses to obtain one pellet. The animals also received control surgery and were tested in the operant chamber for two days, one prior to and the other following the CCK-33 treatment. CCK-33 suppressed the number of meals, the total amount of food eaten, and the total duration of time spent eating. However, the size of each meal and the rate of intake were not affected. The CCK effect did not interact with the light-dark phases of diurnal cycle. It appears that a major effect of continuous systemic elevation of CCK-33 is to reduce food intake by prolonging the satiety period rather than by decreasing the individual meal size.