Biology subjects

Czubala, M. A.

Publications and source records attributed to Czubala, M. A..

3 recordsLinked to original sources

bioRxiv2026

Comprehensive lipidomics of tissue macrophages reveal LTE4-driven eosinophil survival

Tissue-resident peritoneal macrophages (pM[FE]) programmed by GATA6 are essential players regulating immunity and tissue balance in the peritoneal cavity. How GATA6 regulates global lipid metabolism is currently unknown. Addressing this, in myeloid-restricted Gata6-deficient (Gata6-KOmye) mice, significant changes to the pM[FE] lipidome were found. First, Bodipy staining and anti-Stokes Raman scattering (CARS) microscopy demonstrated significant intracellular lipid accumulation in lipid droplets in Gata6-KOmye. Untargeted and targeted lipidomics revealed this to result from increased levels of multiple sphingolipid (SL) molecular species, including sphingomyelins, ceramides, and glycosphingolipids, along with upregulation of the cysteinyl leukotriene (CysLTs) pathway at both lipidomic and transcriptional levels. Evidencing a functional role for the lipidomic phenotype, Gata6-KOmye showed significant eosinophil accumulation, associated with decreased apoptosis which was exclusively driven by CysLT signalling. In summary, GATA6 is demonstrated as a regulator of sphingolipid accumulation and CysLT generation in pM[FE], with secondary impacts on associated leukocytes through regulation of transcellular CysLT signaling.

immunology↗

bioRxiv2026

Highly Efficient Lentiviral Transduction of Human iPSC-Derived Microglia and Macrophages

BackgroundMicroglia have become a cell type of interest in the neurodegenerative field given both genetic and pathological evidence for their role in disease development and progression. There has been a rapid growth of studies using iPSC-derived microglial models to understand the molecular mechanisms driving these neurological diseases. However, it remains difficult to transduce myeloid cells effectively which is critical when aiming to study the role of disease associated genes and pathways. Current methods require exposure to multiple viruses which is not suitable for all experimental paradigms. We have therefore sought and characterised a high efficiency promoter and plasmid design to allow high transduction efficacy with a single lentivirus. ResultsUsing the spleen focus-forming virus (SFFV) promoter in combination with central polypurine tract (cPPT) and Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) plasmid elements gave significantly higher transduction efficiency and transgene expression than was achieved with commonly used promoters CMV and EF1. This could then be further improved if required to over 90% transduction efficiency with the removal of lentivirus restriction factor SAM and HD domain-containing protein 1 (SAMHD1) by adding VPX. ConclusionsOur findings allow for a simpler, more efficient and streamlined approach to transgene expression in iPSC-derived microglia and macrophages using only a single lentivirus. This minimises potential unintended side effects such as additional cellular activation and increased cell death.

neuroscience↗

bioRxiv2020

The Alzheimer's disease protective P522R variant of PLCG2, consistently enhances stimulus-dependent PLCγ2 activation, depleting substrate and altering cell function.

Recent genome-wide association studies of Alzheimers disease (AD) have identified variants implicating immune pathways in disease development. A rare coding variant of PLCG2, which encodes PLC{gamma}2, shows a significant protective effect for AD (rs72824905, P522R, P=5.38x10-10, Odds Ratio = 0.68). Molecular dynamic modelling of the PLC{gamma}2-R522 variant, situated within the auto-inhibitory domain of PLC{gamma}2, suggests a structural change to the protein. Through CRISPR-engineering we have generated novel PLCG2-R522 harbouring human induced pluripotent cell lines (hiPSC) and a mouse knockin model, neither of which exhibits alterations in endogenous PLCG2 expression. Mouse microglia and macrophages and hiPSC-derived microglia-like cells with the R522 mutation, all demonstrate a consistent non-redundant hyperfunctionality in the context of normal expression of other PLC isoforms. This signalling alteration manifests as enhanced cellular Ca2+ store release ([~]20-40% increase) in response to physiologically-relevant stimuli (e.g. Fc receptor ligation and A{beta} oligomers). This hyperfunctionality resulted in increased PIP2 depletion in the cells with the PLC{gamma}2-R522 variant after exposure to stimuli and reduced basal detection of PIP2 levels in vivo. These PLC{gamma}2-R522 associated abnormalities resulted in impairments to phagocytosis (fungal and bacterial particles) and enhanced endocytosis (A{beta} oligomers and dextran). PLC{gamma}2 sits downstream of disease relevant pathways, such as TREM2 and CSF1R and alterations in its activity, direct impacts cell function, which in the context of the inherent drugability of enzymes such as PLC{gamma}2, raise the prospect of manipulation of PLC{gamma}2 as a therapeutic target in Alzheimers Disease.

neuroscience↗