Biology subjects

Savulescu, A. F.

Publications and source records attributed to Savulescu, A. F..

6 recordsLinked to original sources

bioRxiv2025

Protein Kinase C δ: a critical hub regulating macrophage immunomodulatory functions during Mycobacterium tuberculosis infection

A host-modulating candidate gene involved in putative pathogen-killing pathways, with potential novel therapeutic intervention, Protein Kinase C - {delta} (PKC{delta}) has been recognized as a critical marker of inflammation with clinical and experimental evidence in recent years. Pulmonary microenvironment during Mtb infection is largely governed by lung resident macrophages, initiating innate and subsequent adaptive immune responses. We investigated the role of PKC{delta} in macrophages using a macrophage-specific PKC{delta} knockout mice model (LysMcrePKC{delta}flox/flox). PKC{delta} deficiency in macrophages triggers an early lymphocytic immune response, increases neutrophil recruitment, and reduces inflammatory macrophages in the lungs, leading to higher Mtb burden and exacerbated pathology. Experimental and omics analysis further revealed that dysregulation of antimicrobial effector functions is detrimental to macrophages ability to restrict bacterial growth in vitro. Importantly this defect was mitigated by exogenous GM-CSF supplementation and/or overexpressing PKC{delta} in macrophages. Thus, PKC{delta} plays a crucial role in immune modulation during Mtb infection with GM-CSF amongst several downstream pathways through which PKC{delta} exerts its regulatory effects. TeaserPKC{delta} is crucial for immune modulation during Mtb infection revealing macrophages as a potential axis of signaling.

immunology↗

bioRxiv2023

Immunoglobulin M regulates airway hyperresponsiveness independent of T helper 2 allergic inflammation.

Allergic asthma is a disease driven by T helper 2 (Th2) cells, eosinophilia, airway hyperresponsiveness (AHR) and IgE-secreting B cells. Asthma is largely controlled by corticosteroids and {beta}2 adregenic receptor agonists that target and relax airway smooth muscle (ASM). Immunoglobulin M (IgM) isotype secreted by naive B cells is important for class switching but may have other undefined functions. We investigated the role of IgM in a house dust mite (HDM)-induced Th2 allergic asthma model. We sensitised wild-type (WT) and IgM-deficient (IgM KO) mice with HDM and measured AHR, and Th2 responses. We performed RNA sequencing on the whole lung of WT and IgM KO mice sensitised to saline or HDM. We validated our AHR data on human ASM by deleting genes using CRISPR and measuring contraction by single-cell force cytometry. We found IgM to be essential in AHR but not Th2 airway inflammation or eosinophilia. RNA sequencing of lung tissue suggested that IgM regulated AHR through modulating brain-specific angiogenesis inhibitor 1-associated protein 2-like protein 1 (Baiap2l1) and other genes. Deletion of BAIAP2L1 led to a differential reduction in human ASM contraction when stimulated with TNF- and Acetylcholine, but not IL-13. These findings have implications for future treatment of asthma beyond current therapies.

immunology↗

bioRxiv2022

Quantifying spatial dynamics and regulators of Mycobacterium tuberculosis phagocytosis by primary human macrophages using microfabricated patterns

Macrophages provide a first line of defense against invading pathogens, including the leading cause of bacterial mortality, Mycobacterium tuberculosis (Mtb). Phagocytosing extracellular organisms mediate pathogen clearance via a multitude of antimicrobial mechanisms, uniquely designed against an array of pathogens. Macrophages are able to execute different programs of activation in response to pathogenic challenge with host mediators, polarizing them to a variety of differentiation states, including the pro-inflammatory M1 and anti-inflammatory M2 states. The functional polarization of a macrophage prior to infection, thus impacts the outcome of host-pathogen interaction. One of the limitations when using in vitro differentiated human primary monocyte-derived macrophages (MDMs) is the heterogeneous nature of the mature population, which presents a challenge for quantitative characterization of various host-pathogen processes. Here, we describe an approach to minimize this heterogeneity, based on micropatterning of cells to reintroduce aspects of cellular homogeneity lost in a 2D tissue culture. Micropatterning consists of growing cells at the single cell level on microfabricated patterns, to constrain the size and shape of the cell, reducing cell-to-cell variation and mimicking the physiological spatial confinement that cells experience in tissues. We infected micropatterned GM-CSF- (M1) and M-CSF- (M2) derived human MDMs with Mtb, which allowed us to study host-pathogen interactions at a single cell level, at high resolution and in a quantitative manner, across tens to hundreds of cells in parallel. Using our approach, we were able to quantify phagocytosis of Mtb in MDMs, finding phagocytic contraction is increased by interferon-gamma stimulation, whilst contraction and bacterial uptake is decreased following silencing of phagocytosis regulator NHLRC2 or Tween80 removal of bacterial surface lipids. We also identify alterations in host organelle position within Mtb infected MDMs, as well as identifying differences in Mtb subcellular localization in relation to the microtubule organizing center (MTOC) and in line with the cellular polarity in M1 and M2 MDMs. Our approach described here can be adapted to study other host-pathogen interactions and co-infections in MDMs and can be coupled with downstream automated analytical approaches.

cell biology↗

bioRxiv2021

DeepSpot: a deep neural network for RNA spot enhancement in smFISH microscopy images

Detection of RNA spots in single molecule FISH microscopy images remains a difficult task especially when applied to large volumes of data. The small size of RNA spots combined with high noise level of images often requires a manual adaptation of the spot detection thresholds for each image. In this work we introduce DeepSpot, a Deep Learning based tool specifically designed to enhance RNA spots which enables spot detection without need to resort to image per image parameter tuning. We show how our method can enable the downstream accurate detection of spots. The architecture of DeepSpot is inspired by small object detection approaches. It incorporates dilated convolutions into a module specifically designed for the Context Aggregation for Small Object (CASO) and uses Residual Convolutions to propagate this information along the network. This enables DeepSpot to enhance all RNA spots to the same intensity and thus circumvents the need for parameter tuning. We evaluated how easily spots can be detected in images enhanced by our method, by training DeepSpot on 20 simulated and 1 experimental datasets, and have shown that more than 97% accuracy is achieved. Moreover, comparison with alternative deep learning approaches for mRNA spot detection (deepBlink) indicated that DeepSpot allows more precise mRNA detection. In addition, we generated smFISH images from mouse fibroblasts in a wound healing assay to evaluate whether DeepSpot enhancement can enable seamless mRNA spot detection and thus streamline studies of localized mRNA expression in cells.

bioinformatics↗

bioRxiv2021

Mitochondria serve as axonal shuttle for Cox7c mRNA through mechanism that involves its mitochondrial targeting signal

Localized protein synthesis plays a key role in spatiotemporal regulation of the cellular proteome. Neurons, which extend axons over long distances, heavily depend on this process. However, the mechanisms by which axonal mRNAs are transported to protein target sites are not fully understood. Here, we describe a novel role for mitochondria in shuttling a nuclear encoded mRNA along axons. Fractionation analysis and smFISH revealed that the mRNA encoding Cox7c protein is preferentially associated with mitochondria from a neuronal cell line and from primary motor neuron axons. Live cell imaging of MS2-tagged Cox7c or Cryab control mRNA in primary motor neurons further confirmed the preferential colocalization of Cox7c mRNA with mitochondria. More importantly, Cox7c demonstrated substantial cotransport with mitochondria along axons. Intriguingly, the coding region, rather than the 3UTR, was found to be the key domain for the cotransport. Furthermore, we show that puromycin treatment as well as hindering the synthesis of the mitochondrial targeting signal (MTS) reduced the colocalization. Overall, our results reveal a novel mRNA transport mode which exploits mitochondria as a shuttle and translation of the MTS as a recognition feature. Thus, mitochondria may play a role in spatial regulation of the axonal transcriptome and self-sustain their own proteome at distant neuronal sites.

cell biology↗

bioRxiv2019

DypFISH: Dynamic Patterned FISH to Interrogate RNA and Protein Spatial and Temporal Subcellular Distribution

Advances in single cell RNA sequencing have allowed for the identification and characterization of cellular subtypes based on quantification of the number of transcripts in each cell. However, cells may differ not only in the number of mRNA transcripts that they exhibit, but also in their spatial and temporal distribution, intrinsic to the definition of their cellular state. Here we describe DypFISH, an approach to quantitatively investigate the spatial and temporal subcellular localization of RNA and protein, by combining micropatterning of cells with fluorescence microscopy at high resolution. We introduce a range of analytical techniques for quantitatively interrogating single molecule RNA FISH data in combination with protein immunolabeling over time. Strikingly, our results show that constraining cellular architecture reduces variation in subcellular mRNA and protein distributions, allowing the characterization of their localization and dynamics with high reproducibility. Many tissues contain cells that exist in similar constrained architectures. Thus DypFISH reveals reproducible patterns of clustering, strong correlative influences of mRNA-protein localization on MTOC orientation when they are present and interdependent dynamics globally and at specific subcellular locations which can be extended to physiological systems.

systems biology↗