Biology subjects

Kubatzky, K.

Publications and source records attributed to Kubatzky, K..

3 recordsLinked to original sources

bioRxiv2022

Gαq modulates the energy metabolism of osteoclasts

The bacterial protein toxin Pasteurella multocida toxin (PMT) mediates RANKL-independent osteoclast differentiation. Although these osteoclasts are small, their resorptive activity is high and destroys the nasal turbinate bones of pigs. Analysis of the proteome of classical and toxin-derived osteoclasts showed that PMT induces the upregulation of metabolic pathways. This includes strong glycolytic activity, increased expression of GLUT1 and upregulation of the mTOR pathway. As OxPhos components are also expressed more efficiently, cells display increased mitochondrial respiration. We found that the heterotrimeric G protein Gq plays a central role in this hypermetabolic cell activation. Gq triggers mitochondrial relocalisation of pSerSTAT3 and an increase in OPA1 expression. Overexpression of Gq in Hoxb8 cells mimicked this hypermetabolic phenotype and resulted in higher glycolytic and mitochondrial activity as well as increased bone resorptive activity. Rheumatoid arthritis patients show an increase in Gnaq expression especially in the synovial fluid, suggesting that Gq is a target of pathophysiological relevance.

immunology↗

bioRxiv2022

Acod1 negatively impacts osteoclastogenesis via GPR91-mediated NFATc1 activation

Immune cells undergo metabolic reprogramming upon exposure to inflammatory stimuli. The immuneresponsive gene 1 (Irg1) encodes aconitate decarboxylase (Acod1), an enzyme that generates itaconate from cis-aconitate in the TCA cycle and is one of the most highly induced genes in macrophages during inflammation. Itaconate inhibits succinate dehydrogenase activity leading to the accumulation of succinate. As the adjustment of energy metabolism also plays an important role during the formation of bone-resorbing osteoclasts, we investigated if Irg1 is regulated during osteoclastogenesis. We show that M-CSF/RANKL treatment induces Irg1 at an early time-point in bone marrow-derived macrophages (BMDM) as well as in the RAW264.7 macrophage cell line. Next, we stably overexpressed Acod1 in RAW264.7 cells. The metabolism of these Acod1 cells shifted towards glycolysis, as indicated by an increase in mTOR activation, subsequent 4EB-P1 phosphorylation and reduced ATP levels. When we investigated the ability of Acod1 cells to differentiate into osteoclasts, we observed a remarkable suppression of osteoclast-associated genes and the number of TRAP-positive, multi-nucleated osteoclasts was greatly reduced but not completely abrogated. Surprisingly, NFATc1 was detectable in nuclear extracts in untreated Acod1 cells leading to residual transcriptional NFAT activity in luciferase assays. This is caused by the elevated levels of succinate in Acod1 cells, as succinate can bind extracellularly to its cognate receptor GPR91 leading to Gq-mediated activation of NFATc1. When we investigated the expression of Gpr91, we found RANKL-mediated induction of Gpr91 to be severely reduced in Acod1 cells and we suggest that GPR91 is a target of RANKL-mediated NFATc1 activation. However, on the protein level, the receptor was still expressed at the cell surface. The observed repression of Gpr91 in Acod1 overexpressing cells was also detected by treatment with octyl-itaconate, showing that this is an itaconate-mediated effect. We hypothesize that the itaconate-mediated increase in succinate levels causes activation of NFATc1 signalling, although the transcriptional activity does not lead to osteoclastogenesis. In the presence of RANKL, these pre-activated cells are slow in switching to RANKL-mediated induction of osteoclast genes, which decreases their ability to differentiate into osteoclasts.

immunology↗

bioRxiv2021

Staphylococci planktonic and biofilm environments differentially affect macrophage immune activation and osteoclastogenic differentiation.

Biofilm formation is a leading cause for chronic implant-related bone infections as biofilms shield bacteria against the immune system and antibiotics. Additionally, biofilms generate a metabolic microenvironment that shifts the immune response towards tolerance. Here, we compared the impact of the metabolite profile of bacterial environments on macrophage immune activation using Staphylococcus aureus (SA) and epidermidis (SE) conditioned media (CM) of planktonic and biofilm cultures. The biofilm environment had reduced glucose and increased lactate concentrations. Moreover, the expression of typical immune activation markers on macrophages was reduced in the biofilm environment compared to the respective planktonic CM. However, all CM caused a predominantly pro-inflammatory macrophage cytokine response with a comparable induction of Tnfa expression. In biofilm CM this was accompanied by higher levels of anti-inflammatory Il10. Planktonic CM on the other hand, induced an IRF-7 mediated Ifnb expression which was absent in the biofilm environments. For SA but not for SE planktonic CM, this was accompanied by IRF3 activation. Stimulation of macrophages with TLR-2/-9 ligands under varying metabolic conditions revealed that, like in the biofilm setting, low glucose concentration reduced the Tnfa to Il10 mRNA ratio. However, addition of extracellular L-lactate but not D-lactate increased the Tnfa to Il10 mRNA ratio upon TLR-2/-9 stimulation. In summary, our data indicate that the mechanisms behind the activation of macrophages differs between planktonic and biofilm environments. These differences are independent of the metabolite profiles, suggesting that the production of different bacterial factors is ultimately more important than the concentrations of glucose and lactate in the environment.

immunology↗