Biology subjects

Philpott, D. J.

Publications and source records attributed to Philpott, D. J..

9 recordsLinked to original sources

bioRxiv2025

Endothelial cell-intrinsic NOD2 signaling regulates the intestinal immune response through the generation of effector and memory T cells.

Crohns disease (CD) is marked by vascular endothelial dysfunction and aberrant T cell immunity in the gastrointestinal tract. However, mechanistic understanding is lacking of how CD-associated gene variants, particularly those that compromise NOD2 function, impact the intestinal vascular endothelium and orchestration of T cell immunity. Here, we find that NOD2, when triggered by its ligand, muramyl dipeptide, is unique among pattern recognition receptors in its ability to induce endothelial cell expression of chemokines and immune adhesion molecules. Consequently, NOD2 signaling promoted T cell homing specifically to gut-associated lymphoid tissue during intestinal infection. Endothelial cell-specific deletion of Nod2 resulted in fewer effector and memory T cells in the small intestine, impacting the hosts ability to clear secondary infection. Together, our findings suggest that vascular endothelial cell expression of NOD2 coordinates intestinal immune responses, and that CD-associated loss of NOD2 function promotes aberrant inflammation due to alterations in the magnitude and specificity of host defense within the intestine.

immunology↗

bioRxiv2024

GSDMD pore formation regulates caspase-4 cleavage to limit IL-18 production in the intestinal epithelium

Epithelial inflammasomes induce pyroptosis and release cytokines to defend against cytosolic pathogens. However, pyroptosis in epithelial barriers must be carefully regulated to facilitate elimination of infected cells while limiting widespread pyroptosis to preserve the single cell barrier. How epithelial cells achieve this is unknown. In this study, we describe a novel epithelial caspase regulation mechanism. By examining caspase-4 activation in human epithelial cells, we discovered that GSDMD pore formation serves as a signal to terminate caspase-4 activity thus facilitating epithelial cell expulsion while controlling cytokine secretion. Inhibition of epithelial pyroptosis led to IL-18 hyperproduction, likely as a mechanism to combat increased pathogen burden and initiate a wider immune response. Moreover, we demonstrate that full-length, rather than cleaved caspase-4 is active against IL-18 and propose that GSDMD pore formation facilitates cleavage of caspase-4 to terminate its catalytic activity. By comparing human cells and murine epithelial organoids to immune cells, we show that GSDMD pore mediated inhibition of caspase activity is largely specific to epithelial cells. Overall, these studies characterise a novel, epithelial-specific negative feedback loop that modulates inflammasome activity and challenge the dogma that autocatalytic caspase cleavage is required for caspase activity against substrates. Graphical AbstractIn intestinal epithelial cells, caspase activation simultaneously leads to GSDMD pore formation and IL-18 release. GSDMD pore formation provides a signal to terminate caspase activity and limit cytokine production. In GSDMD deficient cells, lack of an inhibition signal leads to caspase mediated IL-18 hyperproduction. Upon cell death this leads to release of massive amounts of IL-18. Created with BioRender.com O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/578487v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@46f768org.highwire.dtl.DTLVardef@11cf918org.highwire.dtl.DTLVardef@125be35org.highwire.dtl.DTLVardef@ea9ead_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

Bacterial ADP-heptose initiates a revival stem cell program in the intestinal epithelium

The intestinal epithelium has an exceptional capacity to repair following injury, and recent evidence has suggested that YAP-dependent signaling was crucial for the expansion of Clu+ revival stem cells (revSCs) with fetal-like characteristics, which are essential for epithelial regeneration. However, neither the mechanism underlying where these revSCs emerge from nor the nature of the physiological cues that induce this revSC program, are clearly identified. Here, we first demonstrate that Alpk1 and Tifa, which encode the proteins essential for the detection of the bacterial metabolite ADP-heptose (ADP-Hep), were expressed by the stem cell pool in the intestinal epithelium. Treatment of intestinal organoids with ADP-Hep not only induced acute NF-{kappa}B pro-inflammatory signaling but also TNF-dependent apoptosis within the crypt, causing blunted proliferation and acute disruption of the crypt architecture, while also triggering induction of a revSC program. To identify the molecular underpinnings of this process, we performed single-cell RNA-seq analysis of ADP-Hep-treated organoids as well as lineage-tracing experiments. Our data reveal that ADP-Hep induced the specific ablation of the homeostatic intestinal stem cell (ISC) pool. Removal of ADP-Hep resulted in the rapid recovery of ISCs through dedifferentiation of Paneth cells, which transiently acquired revSC features and expressed nuclear YAP. Moreover, lineage tracing from Lyz1+ Paneth cells showed that ADP-Hep triggered Paneth cell de-differentiation towards pluripotent and proliferative cells in organoids. In vivo, revSC emergence in response to irradiation-induced injury was severely blunted in Tifa-deficient mice, suggesting that efficient epithelial regeneration in this model required detection of microbiota-derived ADP-Hep by the ALPK1-TIFA pathway. Together, our work reveals that Paneth cells can serve as the cell of origin for revSC induction in the physiological context of microbial stimulation, and that the transient loss of Alpk1-expressing ISCs is the initiating event for this regenerative process.

cell biology↗

bioRxiv2023

Cytosolic retention of the mitochondrial protease HtrA2 during mitochondrial protein import stress (MPIS) triggers the DELE1-HRI pathway

Mitochondrial stress inducers, such as the proton ionophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and the ATPase pump inhibitor oligomycin, trigger the DELE1-HRI branch of the integrated stress response (ISR) pathway. Previous studies performed using epitope-tagged forms of DELE1 showed that these stresses induced the accumulation of a cleaved form of DELE1, DELE1-S, which stimulates HRI. Here, we report that mitochondrial protein import stress (MPIS) is an overarching stress that triggers the DELE1-HRI pathway, and that endogenous DELE1 could be cleaved into two forms, DELE1-S and DELE1-VS, the latter accumulating only upon non- depolarizing MPIS. We further showed that DELE1 specifically senses MPIS triggered by the inhibition of the TIM23 complex at the inner mitochondrial membrane (IMM). While MPIS can also cause mitophagy induction through engagement of the NLRX1-RRBP1 pathway, we observed that DELE1-HRI and NLRX1-RRBP1 signaling were engaged independently upon MPIS. Surprisingly, our results suggest that in our cellular model the mitochondrial protease OMA1 was dispensable for DELE1 cleavage upon MPIS. Instead, we identified a key role for another mitochondrial protease, HtrA2, in mediating the cleavage of DELE1 into DELE1-S and DELE1- VS. Our data further suggest that DELE1 is likely cleaved into DELE1-S by HtrA2 in the cytosol, while the DELE1-VS form might be generated during halted translocation of the protein into mitochondria. Together, this study identifies MPIS as the overarching stress detected by DELE1 and identifies HtrA2 as a critical protease involved in DELE1 processing.

cell biology↗

bioRxiv2020

Bone marrow adipocytes drive the development of tissue invasive Ly6Chigh monocytes during obesity

During obesity and high fat-diet (HFD) feeding in mice, sustained low-grade inflammation includes not only increased pro-inflammatory macrophages in the expanding adipose tissue, but also bone marrow (BM) production of invasive Ly6Chigh monocytes. As BM adiposity also accrues with HFD, we explored the relationship between the gains in BM white adipocytes and invasive Ly6Chigh monocytes in vivo and through ex vivo paradigms. We find a temporal and causal link between BM adipocyte whitening and the Ly6Chigh monocyte surge, preceding the adipose tissue macrophage rise during HFD. Phenocopying this, ex vivo treatment of BM cells with conditioned media from BM adipocytes or from bona fide white adipocytes favoured Ly6Chigh monocyte preponderance. Notably, the switch towards the Ly6Chigh subset was preceded by monocyte metabolic reprogramming towards glycolysis, reduced oxidative potential and increased mitochondrial fission. In sum, short-term HFD changes BM cellularity, resulting in local adipocyte whitening driving a gradual increase and activation of invasive Ly6Chigh monocytes.

immunology↗

bioRxiv2020

The eIF2α kinase HRI triggers the autophagic clearance of cytosolic protein aggregates

Large cytosolic protein aggregates are removed by two main cellular processes, autophagy and the ubiquitin-proteasome system (UPS), and defective clearance of these protein aggregates results in proteotoxicity and cell death. Here we show that the eIF2 kinase HRI potentiates the autophagic clearance of cytosolic protein aggregates when the UPS is inhibited. In cells silenced for HRI, proteasome inhibition resulted in accumulation of aggresomes and ubiquitinated proteins, as well as cytotoxicity. Moreover, silencing of HRI resulted in cytotoxic accumulation of over-expressed -synuclein, a protein known to aggregate in Parkinsons disease, dementia with Lewy bodies, and multiple system atrophy. In agreement, protein aggregate accumulation and microglia activation were observed in the spinal cord white matter of 7-month old Hri-/- mice as compared to Hri+/+ littermates. Moreover, aged Hri-/- mice showed accumulation of misfolded -synuclein, indicative of misfolded proteins, in the lateral collateral pathway, a region of the sacral spinal cord horn that receives visceral sensory afferents from the bladder and distal colon, a pathological feature common to -synucleinopathies in humans where it may contribute to impaired micturition and/or constipation. Together, these results suggest that HRI contributes to a general cytosolic unfolded protein response (cUPR) that could be leveraged to bolster the clearance of cytotoxic protein aggregates.

cell biology↗

bioRxiv2019

Nod-like receptors are critical for gut-brain axis signaling

Gut-brain axis signaling is critical for maintaining health and homeostasis. Stressful life events can impact gut-brain signaling, leading to altered mood, cognition and intestinal dysfunction. Here we identify nucleotide binding oligomerization domain (Nod)-like receptors (NLR), Nod1 and Nod2, as novel regulators for gut-brain signaling. NLR are innate immune pattern recognition receptors expressed in the gut and brain, important in the regulation of gastrointestinal (GI) physiology. We found that mice deficient in both Nod1 and Nod2 (NodDKO) demonstrate signs of stress-induced anxiety, cognitive impairment and depression in the context of a hyperactive hypothalamic-pituitary-adrenal axis. These deficits were coupled with impairments in the serotonergic pathway in the brain, decreased hippocampal neurogenesis, and reduced neural activation. In addition, NodDKO mice had increased GI permeability and altered serotonin signaling in the gut following exposure to acute stress. Administration of the selective serotonin reuptake inhibitor, fluoxetine, abrogated behavioral impairments and restored serotonin signaling. We also identified that intestinal epithelial cell-specific deletion of Nod1 (VilCre+Nod1f/f), but not Nod2, increased susceptibility to stress-induced anxiety-like behavior and cognitive impairment following exposure to stress. Together these data suggest that intestinal epithelial NLR are novel modulators of gut-brain communication and may serve as potential novel therapeutic targets for the treatment of gut-brain disorders.

physiology↗

bioRxiv2019

NOD1 mediates non-canonical inflammasome processing of interleukin-18 in epithelial cells to Helicobacter pylori infection.

The interleukin-1 family members, IL-1{beta} and IL-18, are processed into their biologically active forms by multi-protein complexes, known as inflammasomes. Although the inflammasome pathways that mediate IL-1{beta} processing in myeloid cells have been extensively studied, those involved in IL-18 processing, particularly in non-myeloid cells, are still poorly understood. Here, we have identified the cytosolic sensor NOD1 as a key regulator of IL-18 processing in epithelial cells responding to Helicobacter pylori infection. Importantly, NOD1 processing of IL-18 occurs independently of the canonical inflammasome proteins, NLRP3 and ASC. Instead, NOD1 interacts directly with caspase-1 via homotypic binding of caspase-activation recruitment domains. We show that IL-18 is important in maintaining tissue homeostasis and protecting against pre-neoplastic changes due to gastric H. pylori infection. These findings reveal an unanticipated role for NOD1 in a new type of inflammasome that regulates epithelial cell production of bioactive IL-18 with tissue protective functions.

immunology↗

bioRxiv2019

Trace levels of peptidoglycan in serum underlie the NOD-dependent cytokine response to endoplasmic reticulum stress

NOD1 and NOD2 are intracellular sensors of bacterial peptidoglycan that belong to the Nod-like receptor (NLR) family of innate immune proteins. In addition to their role as direct bacterial sensors, it was recently proposed that NOD proteins could detect endoplasmic reticulum (ER) stress induced by thapsigargin, an inhibitor of the sarcoplasmic or endoplasmic reticulum calcium ATPase family (SERCA) that pumps Ca2+ into the ER, resulting in pro-inflammatory signalling. Here, we confirm that thapsigargin induces pro-inflammatory signalling in epithelial cells in a NOD-dependent manner. However, the effect was specific to thapsigargin, as tunicamycin and the subtilase cytotoxin SubAB from Shiga toxigenic Escherichia coli, which induce ER stress by other mechanisms, did not induce cytokine expression. The calcium ionophore A23187 also induced NOD-dependent signalling, and the calcium chelator BAPTA-AM blunted thapsigargin-dependent pro-inflammatory signalling, showing NOD proteins responded to a rise in intracellular Ca2+. Since intracellular Ca2+ directly affects vesicular trafficking, we tested if thapsigargin-induced NOD activation required endocytosis. Our results demonstrate that both endocytosis and the addition of serum to the cell culture medium were required for thapsigargin-mediated NOD activation. Finally, we analyzed cell culture grade fetal calf serum as well as serum from laboratory mice by high-pressure liquid chromatography and mass spectrometry, and identified the presence of various peptidoglycan fragments. We propose that cellular perturbations that affect intracellular Ca2+ can trigger internalization of peptidoglycan trace contaminants found in culture serum, thereby stimulating pro-inflammatory signalling. The presence of peptidoglycan in animal serum suggests that a homeostatic function of NOD signalling may have been previously overlooked.

immunology↗