The NLRP3 inflammasome is activated in response to microbial and danger signals, resulting in caspase-1–dependent secretion of the proinflammatory cytokines IL-1β and IL-18. Canonical NLRP3 inflammasome activation is a two-step process requiring both priming and activation signals. During inflammasome activation, NLRP3 associates with mitochondria; however, the role for this interaction is unclear. In this article, we show that mouse NLRP3 and caspase-1 independently interact with the mitochondrial lipid cardiolipin, which is externalized to the outer mitochondrial membrane at priming in response to reactive oxygen species. An NLRP3 activation signal is then required for the calcium-dependent association of the adaptor molecule ASC with NLRP3 on the mitochondrial surface, resulting in inflammasome complex assembly and activation. These findings demonstrate a novel lipid interaction for caspase-1 and identify a role for mitochondria as supramolecular organizing centers in the assembly and activation of the NLRP3 inflammasome.

Epithelial and stromal/mesenchymal limbal stem cells contribute to corneal homeostasis and cell renewal. Extracellular vesicles (EVs), including exosomes (Exos), can be paracrine mediators of intercellular communication. Previously, we described cargos and regulatory roles of limbal stromal cell (LSC)-derived Exos in non-diabetic (N) and diabetic (DM) limbal epithelial cells (LECs). Presently, we quantify the miRNA and proteome profiles of human LEC-derived Exos and their regulatory roles in N- and DM-LSC. We revealed some miRNA and protein differences in DM vs. N-LEC-derived Exos’ cargos, including proteins involved in Exo biogenesis and packaging that may affect Exo production and ultimately cellular crosstalk and corneal function. Treatment by N-Exos, but not by DM-Exos, enhanced wound healing in cultured N-LSCs and increased proliferation rates in N and DM LSCs vs. corresponding untreated (control) cells. N-Exos-treated LSCs reduced the keratocyte markers ALDH3A1 and lumican and increased the MSC markers CD73, CD90, and CD105 vs. control LSCs. These being opposite to the changes quantified in wounded LSCs. Overall, N-LEC Exos have a more pronounced effect on LSC wound healing, proliferation, and stem cell marker expression than DM-LEC Exos. This suggests that regulatory miRNA and protein cargo differences in DM- vs. N-LEC-derived Exos could contribute to the disease state.

This Technical Note presents a comprehensive proteomics workflow for the new combination of Orbitrap and Astral mass analyzers across biofluids, cells, and tissues. Central to our workflow is the integration of Adaptive Focused Acoustics (AFA) technology for cells and tissue lysis to ensure robust and reproducible sample preparation in a high-throughput manner. Furthermore, we automated the detergent-compatible single-pot, solid-phase-enhanced sample Preparation (SP3) method for protein digestion. The synergy of these advanced methodologies facilitates a robust and high-throughput approach for cell and tissue analysis, an important consideration in translational research. This work disseminates our platform workflow, analyzes the effectiveness, demonstrates the reproducibility of the results, and highlights the potential of these technologies in biomarker discovery and disease pathology. For cells and tissues (heart, liver, lung, and intestine) proteomics analysis by data-independent acquisition mode, identifications exceeding 10,000 proteins can be achieved with a 24 min active gradient. In 200 ng injections of HeLa digest across multiple gradients, an average of more than 80% of proteins have a CV less than 20%, and a 45 min run covers ∼90% of the expressed proteome. This complete workflow allows for large swaths of the proteome to be identified and is compatible with diverse sample types.

Almost 30% of survivors of myocardial infarction (MI) develop heart failure (HF), in part due to damage caused by the accumulation of dysfunctional mitochondria. Organelle quality control through Parkin-mediated mitochondrial autophagy (mitophagy) is known to play a role in mediating protection against HF damage post-ischaemic injury and remodelling of the subsequent deteriorated myocardium. This study has shown that a single i.p. dose (2 h post-MI) of the selective small molecule Parkin activator PR-364 reduced mortality, preserved cardiac ejection fraction, and mitigated the progression of HF. To reveal the mechanism of PR-364, a multi-omic strategy was deployed in combination with classical functional assays using in vivo MI and in vitro cardiomyocyte models. In vitro cell data indicated that Parkin activation by PR-364 increased mitophagy and mitochondrial biogenesis, enhanced adenosine triphosphate production via improved citric acid cycle, altered accumulation of calcium localization to the mitochondria, and initiated translational reprogramming with increased expression of mitochondrial translational proteins. In mice, PR-364 administered post-MI resulted in widespread proteome changes, indicating an up-regulation of mitochondrial metabolism and mitochondrial translation in the surviving myocardium. This study demonstrates the therapeutic potential of targeting Parkin-mediated mitophagy using PR-364 to protect surviving cardiac tissue post-MI from progression to HF. Parkin activator proposed therapeutic mechanisms. Inactive Parkin (red) is activated by PR-364; activated Parkin (green) promotes enhanced mitophagy and mito-biogenesis, improves mitochondrial function via increased mitochondrial calcium level and biogenesis, and ultimately leads to limited post-myocardial infarction cardiac remodelling and heart failure. MI, myocardial infarction.

Objective: We aimed to investigate the hypothesis that interferon (IFN)–stimulated gene (ISG) expression in systemic lupus erythematosus (SLE) monocytes is linked to changes in metabolic reprogramming and epigenetic regulation of ISG expression.Methods: Monocytes from healthy volunteers and patients with SLE at baseline or following IFNα treatment were analyzed by extracellular flux analysis, proteomics, metabolomics, chromatin immunoprecipitation, and gene expression. The histone demethylases KDM6A/B were inhibited using glycogen synthase kinase J4 (GSK‐J4). GSK‐J4 was tested in pristane and resiquimod (R848) models of IFN‐driven SLE.Results: SLE monocytes had enhanced rates of glycolysis and oxidative phosphorylation compared to healthy control monocytes, as well as increased levels of isocitrate dehydrogenase and its product, α‐ketoglutarate (α‐KG). Because α‐KG is a required cofactor for histone demethylases KDM6A and KDM6B, we hypothesized that IFNα may be driving “trained immune” responses through altering histone methylation. IFNα priming (day 1) resulted in a sustained increase in the expression of ISGs in primed cells (day 5) and enhanced expression on restimulation with IFNα. Importantly, decreased H3K27 trimethylation was observed at the promoters of ISGs following IFNα priming. Finally, GSK‐J4 (KDM6A/B inhibitor) resulted in decreased ISG expression in SLE patient monocytes, as well as reduced autoantibody production, ISG expression, and kidney pathology in R848‐treated BALB/c mice.Conclusion: Our study suggests long‐term IFNα exposure alters the epigenetic regulation of ISG expression in SLE monocytes via changes in immunometabolism, a mechanism reflecting trained immunity to type I IFN. Importantly, it opens the possibility that targeting histone‐modifying enzymes, such as KDM6A/B, may reduce IFN responses in SLE. image

To understand how kidney donation leads to an increased risk of preeclampsia, we studied pregnant outbred mice with prior uninephrectomy and compared them with sham-operated littermates carrying both kidneys. During pregnancy, uninephrectomized (UNx) mice failed to achieve a physiological increase in the glomerular filtration rate and during late gestation developed hypertension, albuminuria, glomerular endothelial damage, and excess placental production of soluble fms–like tyrosine kinase 1 (sFLT1), an antiangiogenic protein implicated in the pathogenesis of preeclampsia. Maternal hypertension in UNx mice was associated with low plasma volumes, an increased rate of fetal resorption, impaired spiral artery remodeling, and placental ischemia. To evaluate potential mechanisms, we studied plasma metabolite changes using mass spectrometry and noted that l-kynurenine, a metabolite of l-tryptophan, was upregulated approximately 3-fold during pregnancy when compared with prepregnant concentrations in the same animals, consistent with prior reports suggesting a protective role for l-kynurenine in placental health. However, UNx mice failed to show upregulation of l-kynurenine during pregnancy; furthermore, when UNx mice were fed l-kynurenine in drinking water throughout pregnancy, their preeclampsia-like state was rescued, including a reversal of placental ischemia and normalization of sFLT1 levels. In aggregate, we provide a mechanistic basis for how impaired renal reserve and the resulting failure to upregulate l-kynurenine during pregnancy can lead to impaired placentation, placental hypoperfusion, an antiangiogenic state, and subsequent preeclampsia.