Faculty Bibliography

OBJECTIVES/OBJECTIVE:The gut microbiome plays a crucial role in regulating systemic immunity and has been implicated in several chronic inflammatory diseases. Intestinal expansions of Ruminococcus gnavus (RG), a dominant gut commensal, correlate with disease flares in lupus nephritis (LN), but the underlying mechanism remains unknown. METHODS:In a Pilot cohort of patients with biopsy-proven LN, subsetted by gut microbiota community, immune status was characterised using bulk-blood RNA sequencing libraries, serum levels of representative host proteins, and levels of immunoglobulin (Ig)G antibodies to the novel lipoglycan (LG) produced by pathogenic RG strains. A Validation LN cohort was evaluated for blood transcriptomic profiles and levels of anti-LG antibodies. In murine models, mechanistic hypotheses were tested after RG gut colonisation or after intraperitoneal injection with an LG preparation, with outcomes determined by transcriptomic analyses, platelet functional readouts, and tissue histology. RESULTS:In a Pilot cohort of patients with LN, RG gut expansions were associated with high-level platelet, neutrophil, and monocyte activation. Serum levels of platelet factor 4 and release of neutrophil extracellular traps (NETs) were significantly higher in patients with high serum IgG antibody against the novel RG-specific LG, a marker of in vivo immune exposure. An LN Validation cohort confirmed these correlates and showed that anti-LG antibodies serve as a surrogate for thromboinflammatory profile in this LN-associated endotype. In mice, gut colonisation with LG-producing RG strains or a single LG injection caused megakaryocytosis and platelet activation; RG colonisation with LG-producing strains induced tubulointerstitial injury with NETosis. In vivo responses to LG toxin were Toll-like receptor 2-dependent. CONCLUSIONS:Gut expansions of the RG pathobiont may contribute to autoimmune pathogenesis through the LG toxin and cause LN flares through thromboinflammatory mechanisms in this previously unrecognised LN endotype.

De novo protein synthesis is required for long-lasting synaptic plasticity and memory, but it comes with a great metabolic cost. In the mammalian brain, it remains unclear which cell types and biological mechanisms are critical for sensing and responding to increased metabolic demand. Here, we demonstrate that microglia, the resident macrophages of the brain, are required for metabolic coupling between endothelial cells, astrocytes, and neurons, which fuels protein synthesis in active neurons. Increasing metabolic demand via a motor task stimulates microglia to secrete the hypoxia-responsive protein CYR61, which increases glucose transporter expression in brain vasculature. Depleting microglia reduces training-induced metabolic fluxes and neuronal protein synthesis, which can be reproduced by blocking CYR61 signaling. Thus, we define a neuroimmune metabolic circuit that is required for on-demand protein synthesis in mouse motor cortex.

BACKGROUND:Transmural healing (TMH) indicates resolution of inflammation in all bowel wall layers and is an emerging therapeutic target in Crohn's disease (CD). Standardized sonographic criteria for TMH and early improvement, termed Transmural Response (TMR), have not been established. This systematic review synthesizes published definitions to provide an up-to-date overview of the current evidence base for intestinal ultrasound (IUS)-based assessment in CD. METHODS:This systematic review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Comprehensive searches of databases identified full-text articles that pre-specified TMH, TMR or normal/abnormal bowel on trans-abdominal IUS in pediatric or adult participants with CD. Definitions were summarized descriptively. RESULTS:Eighty-three full-text studies (8033 patients) met eligibility criteria; 39 (47%) defined TMH and 22 (27%) defined TMR. TMH definitions most often included bowel-wall thickness (BWT) ≤ 3mm (31/39, 79%), absent or minimal Doppler flow (25/39, 64%), and preserved bowel wall stratification (10/39, 26%). All TMR definitions required BWT reduction, but thresholds varied (absolute ≥ 1 mm or relative ≥ 25% in 16/22, 73%). Nine studies (9/22, 41%) also required Doppler flow improvement and 4/22 (18%) included additional criteria. Pediatric-specific criteria were reported in 2 TMH and one TMR studies, extrapolating from adult BWT values. Heterogeneity precluded quantitative pooling. CONCLUSIONS:Standardized IUS definitions of TMH and TMR in CD are lacking. Consistent, validated criteria are essential to enable reproducible ultrasound endpoints, support treat-to-target strategies, and facilitate incorporation of IUS into CD clinical trials and routine care.

Among the ways by which oncogenic KRAS upregulates glycolysis in cancer is direct interaction of KRAS4A with hexokinase 1 (HK1), but the mechanism is unknown. HK1 associates with the outer mitochondrial membrane (OMM) where its allosteric regulation depends on homodimerization. Using affinity capture, FRET, and blue native gels, we show that KRAS4A enhances oligomerization of HK1 on the OMM. Modeling the HK1/KRAS4A complex with AlphaFold3 predicts that the membrane association sequences of both HK1 and KRAS4A are oriented toward the OMM. Super-resolution microscopy showed colocalization of HK1 and KRAS4A on the OMM with HK1 enriched at discrete locations. Single-molecule tracking reveals HK1 diffusing freely along the OMM and dwelling at discrete regions where two molecules can be seen to colocalize transiently. KRAS4A expression decreased the diffusion coefficient of HK1 on the organelle. Thus, KRAS4A alters the dynamics of HK1 on the OMM and promotes oligomerization.

BACKGROUND/UNASSIGNED:-deficiency in epicardium-derived cells (EPDCs) contributes to fibro-inflammatory signaling and ACM pathogenesis. METHODS/UNASSIGNED:in cardiomyocytes (Pkp2-cKO), in EPDC (Pkp2-eKO), or in both cardiomyocyte and EPDC (Pkp2-ceKO) via the tissue-specific expression of tamoxifen-inducible Cre recombinase. Nonmyocyte populations were isolated 21 days posttamoxifen injection for single-cell RNA-sequencing. Immunohistochemistry, flow cytometry, quantitative reverse transcription polymerase chain reaction, and echocardiography were used to interrogate cardiac physiology and cellular composition. RESULTS/UNASSIGNED:deletion in cardiomyocytes induced a moderate fibro-inflammatory EPDC phenotype, while deletion in EPDC did not elicit a pathological phenotype, suggesting cardiomyocyte involvement is necessary for ACM pathogenesis. Proinflammatory fibroblasts acquired the senescence-associated secretory phenotype, correlating with elevated senescence associated-βgal staining in the right ventricle. Gene expression, flow cytometry, and histological data also revealed an exaggerated inflammatory response in Pkp2-ceKO mice, which progresses from right to left ventricular predominance. Importantly, macrophages and B cells accumulate in both Pkp2-cKO and Pkp2-ceKO mice compared with controls. Although B-cell depletion delays the early inflammatory and fibrosis response, it did not alter end-stage cardiac physiology. CONCLUSIONS/UNASSIGNED:

Lysosomal phospholipid degradation produces two types of metabolites, either 2-lysophospholipids with saturated fatty acids in sn-1 position or 1-lysophospholipids with unsaturated fatty acids in sn-2 position. They may either be degraded further or re-used for phospholipid synthesis. We found that LPLAT7 (LPGAT1), an acyltransferase of the endoplasmic reticulum, re-acylates specifically lysosome-derived 1-lysophospholipids that carry an unsaturated chain. The enzymatic activity of LPLAT7 was specific for stearoyl-CoA and 1-lyso-2-acyl positional isomers of unsaturated lysophospholipids. In Huh7 cells, Lplat7 knockout prevented the reacylation of 1-lysophospholipids generated by the lysosomal degradation of exogenous ²H-phosphatidylcholine. Inhibition of lysosomal phospholipid degradation reduced the abundance of 1-stearoyl-2-unsaturated PC in Huh7 cells. Lplat7 knockout blunted the loss of unsaturated lysophosphatidylcholine (LPC) in response to lysosomal inhibition, suggesting that LPLAT7 consumes unsaturated LPC formed by lysosomes. In mice, Lplat7 knockout increased the concentration of unsaturated lysophospholipids, reduced the abundance of 1-stearoyl-2-unsaturated species of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine, and inhibited the regeneration of cellular membranes. It also triggered the accumulation of triglycerides, confirming earlier reports that unsaturated lysophospholipids induce lipid droplet formation. Thus, by re-acylating unsaturated 1-lysophospholipids, LPLAT7 shifts lipid metabolism from the biogenesis of lipid droplets to the biogenesis of membranes.

A deficit in dietary protein elicits a nutrient-specific appetite, yet the underlying mechanisms remain poorly understood. In this work, we identify coordinated neuronal and systemic mechanisms in Drosophila that drive an essential amino acid (EAA)-specific appetite. EAA deprivation increases neuropeptide CNMamide (CNMa) expression in gut enterocytes, activating enteric neurons and ellipsoid body neurons in the brain to promote EAA intake through two complementary pathways: a rapid neuronal gut-brain axis and a slower hormonal route. CNMa suppresses the activity of sugar-sensing diuretic hormone 44 (DH44) neurons, thereby reducing carbohydrate intake and biasing feeding toward EAAs. Similarly, protein deprivation in mice promotes an EAA-specific appetite independently of fibroblast growth factor 21 (FGF21). Together, these findings reveal multilayered gut-brain mechanisms that regulate nutrient-specific feeding and maintain EAA homeostasis across species.

BACKGROUND:Exercise links with improved cancer outcomes following a diagnosis of primary breast cancer but experimental evidence and molecular mechanistic interrogation from preclinical studies are limited. The purpose of this study was to evaluate the effects, and dose‒response, of exercise in mouse models of breast cancer, as well as elucidate cancer cell extrinsic and intrinsic responses. METHODS:Independent in vivo modeling was used to investigate the effects of exercise across distinct breast cancer models. Unbiased transcriptomic and metabolomic analyses, alongside cellular and proteomic interrogation, were used to determine tumor microenvironment (TME)- and cancer cell-specific effects. RESULTS:Exercise inhibited breast cancer growth and metastasis across multiple syngeneic mouse models compared to sham control. Tumor growth inhibition was independent of estrogen receptor status, and in the 4T1 model, exercise exerted non-dose-dependent effects. In the Met1 model, exercise decreased TME immune cell content, particularly tumor-associated macrophages, while promoting an activated anticancer innate immune cell gene signature. Concurrent in vivo cancer cell-intrinsic effects were characterized by broad transcriptomic reprogramming including downregulation of metabolic pathways and upregulation of pathways regulating proliferation and apoptosis. Whole tumor metabolomic analyses unveiled broad shifts including decreased nicotinamide adenine dinucleotide (NAD+) and lactate, as well as availability of biosynthetic precursors. Finally, in silico analyses identified TME ligands, such as High Mobility Group Box 2 (HMGB2) and Cardiotrophin-1 (CTF1) as candidate drivers of downstream gene expression changes in cancer cells. CONCLUSION/CONCLUSIONS:Exercise suppresses breast cancer progression, which occurs in conjunction with broad reprogramming of immune TME-cancer processes and their interaction.

Reactivation of quiescent neural stem cells (NSCs) in the central nervous system (CNS) is a tightly controlled process that generates new neurons and glia to maintain homeostasis or enable repair post-injury, but it remains unclear if reactivation of distinct NSC populations is coupled. Here, we discovered that NSC quiescence exit in Drosophila follows a hierarchical sequence, whereby activation of anterior stem cells in the brain lobes precedes and is required for the timely state-transition of more posterior NSCs in the ventral nerve cord. To achieve this, quiescent NSCs transiently activate neuronal genes. This transient neuronal state is temporary and specific to NSC dormancy, as neuronal genes are switched off after stem cells resume proliferation. Blocking neuronal firing in brain lobe neurons delays the onset of posterior NSC reactivation. Our results reveal long-range communication between quiescent NSCs to coordinate reactivation across the CNS, enabled by a transient, plastic neuron-like state that allows direct interaction with neuronal axons.

Cerebral malaria is a major complication of Plasmodium falciparum infection that occurs upon the sequestration of infected red blood cells (iRBCs) in brain capillaries, resulting in the loss of endothelial barrier integrity, brain swelling, and frequently long-term sequelae or death. P. falciparum-iRBCs cause the disruption of human brain microvascular endothelial cell barrier integrity in vitro, mimicking the microenvironment of cerebral malaria, yet the specific mechanisms mediating this process remain unknown. Upon infection of the host RBCs, P. falciparum produces hemozoin, a crystal form of heme generated following the degradation of hemoglobin by the parasite. Here, we show that the endothelial barrier-disrupting activity is found entirely in the hemozoin fraction of P. falciparum-iRBCs. This activity is not caused by the hemozoin crystal itself, which is not able to induce barrier disruption, but by the biomolecules that are associated with it. Treatment of purified P. falciparum hemozoin with proteases inhibits the disruption of endothelial barrier integrity caused by the hemozoin, indicating an important role for proteins in the disruption of the barrier. Conversely, treatment with nucleases did not affect hemozoin barrier-disrupting activity. These results identify a key molecular mechanism in the P. falciparum-mediated brain endothelial barrier disruption during cerebral malaria and may open new avenues for the treatment of this complication.IMPORTANCEWhile several specific biomolecules have been proposed to contribute to the disruption of endothelial barrier integrity in cerebral malaria, no single Plasmodium falciparum- or host-derived factor has been definitively identified as the primary driver of this disruption. Here, we identify the brain endothelial barrier-disruptive P. falciparum-infected red blood cell (iRBC)-derived activity to be caused by biomolecules bound to hemozoin, identifying a key, novel mechanism in the pathogenesis of cerebral malaria. The finding that P. falciparum hemozoin also disrupts a pulmonary endothelial cell barrier opens the possibility that this mechanism underlies other severe malaria complications. The implication of P. falciparum-iRBC-derived proteins in this mechanism is in line with previous reports, providing a novel interpretation of these findings in the context of hemozoin-binding. This knowledge provides a new perspective in the search for specific molecules and mechanisms involved in barrier disruption, which may lead to the development of much-needed specific treatments for cerebral malaria.