Cooper, Shawna A

Background: Alcohol-associated hepatitis (AH) is a severe inflammatory liver condition driven by dysregulated immune responses. CD8 T cells accumulate in the liver during AH, yet their functional role remains poorly defined. Methods: A murine NIAAA chronic-binge model with lipopolysaccharide (LPS) challenge was used. Single-cell RNA sequencing of liver and blood immune cells defined CD8 T cell subsets, followed by pathway analysis and reanalysis of human AH datasets. Spatial relationships were validated by immunohistochemistry of human AH liver tissue with CD8 T RM and liver sinusoidal endothelial cell (LSEC) co-staining. LSEC–CD8 T cell interactions were examined using in vitro co-culture, and signaling pathways were assessed by bulk RNA sequencing and phospho-protein analyses. IL15 function was evaluated by in vivo neutralization. Results: Single-cell RNA sequencing in the murine AH model revealed a distinct population of CD8 tissue-resident memory T cells (T RM ) with heightened activation and proinflammatory cytokine production. Analysis of human AH liver single-cell RNA sequencing data, along with immunohistochemistry validation, supported CD8 T RM enrichment in diseased tissue. IL15 emerged as a prominent pathway linked to T RM activation, and IL15 blockade reduced T RM abundance and attenuated EtOH/ lipopolysaccharide-induced liver injury. Mechanistically, LSECs not only provided a structural niche for T RM retention but also amplified IL15 signalling. In vitro, coculture experiments demonstrated that LSECs intensified activation in pre-stimulated CD8 T cells in a stimulus-dependent manner: under IL15 stimulation, LSECs boosted effector function without inducing cell death, whereas under T-cell receptor stimulation, LSECs drove hyperactivation and activation-induced cell death. Bulk RNA-seq and phospho-protein analysis identified the PI3K-AKT pathway as a shared pathway enhanced by LSEC coculture in activated CD8 T cells. These findings define a context-dependent mechanism in which LSECs promote IL15-driven signaling through AKT pathway amplification, promoting T RM persistence and inflammatory activity in AH. Conclusions: IL15-associated signaling within the hepatic microenvironment, shaped by LSEC-CD8 T-cell interactions, promotes activation and persistence of CD8 T RM cells in AH. Targeting the IL15-LSEC-AKT axis may disrupt pathogenic T RM niches and represent a promising therapeutic strategy for severe AH.

Liver macrophages are central in maintaining hepatic homeostasis and mediating immune responses during liver injury, including fibrosis. Macrophages may have proinflammatory or antiinflammatory properties, but which properties influence fibrosis remains unclear. To explore the role of macrophages in liver fibrosis, we performed single-cell RNA-seq in a mouse model of liver injury and found that macrophage diversity was increased. Marco was among the most significantly upregulated genes, and a population of Marco hi macrophages increased with injury and spatially segregated to nonfibrotic areas. The macrophage receptor with collagenous structure (MARCO) protein is a scavenger receptor expressed by specific subsets of macrophages, and its role in liver fibrosis is unclear. In vitro induction of Marco in bone marrow–derived macrophages decreased proinflammatory gene expression, increased antiinflammatory and antifibrotic gene expression, and enhanced phagocytosis, indicating a restorative phenotype. Adoptive transfer of MARCO + macrophages in a mouse model of liver fibrosis reduced the expression of extracellular matrix–associated (ECM-associated) genes in hepatic stellate cells (HSCs) and reduced collagen deposition, which did not occur with the transfer of MARCO – macrophages. Therefore, MARCO + macrophages have a tissue restorative role in the liver and attenuate fibrogenesis through interaction with HSCs, thereby providing a potential therapeutic pathway for liver fibrosis.

Liver fibrosis is characterized by the activation of perivascular hepatic stellate cells (HSCs), the release of fibrogenic nanosized extracellular vesicles (EVs), and increased HSC glycolysis. Nevertheless, how glycolysis in HSCs coordinates fibrosis amplification through tissue zone-specific pathways remains elusive. Here, we demonstrate that HSC-specific genetic inhibition of glycolysis reduced liver fibrosis. Moreover, spatial transcriptomics revealed a fibrosis-mediated up-regulation of EV-related pathways in the liver pericentral zone, which was abrogated by glycolysis genetic inhibition. Mechanistically, glycolysis in HSCs up-regulated the expression of EV-related genes such as Ras-related protein Rab-31 ( RAB31 ) by enhancing histone 3 lysine 9 acetylation on the promoter region, which increased EV release. Functionally, these glycolysis-dependent EVs increased fibrotic gene expression in recipient HSC. Furthermore, EVs derived from glycolysis-deficient mice abrogated liver fibrosis amplification in contrast to glycolysis-competent mouse EVs. In summary, glycolysis in HSCs amplifies liver fibrosis by promoting fibrogenic EV release in the hepatic pericentral zone, which represents a potential therapeutic target.

Our understanding of inner ear hair cell ultrastructure has heretofore relied upon two-dimensional imaging; however, serial block-face scanning electron microscopy (SBFSEM) changes this paradigm allowing for three-dimensional evaluation. We compared inner ear hair cells of the apical cristae in myo7aa −/− null zebrafish, a model of human Usher Syndrome type 1B, to hair cells in wild-type zebrafish by SBFSEM to investigate possible ribbon synapse ultrastructural differences. Previously, it has been shown that compared to wild type, myo7aa −/− zebrafish neuromast hair cells have fewer ribbon synapses yet similar ribbon areas. We expect the recapitulation of these results within the inner ear apical crista hair cells furthering the knowledge of three-dimensional ribbon synapse structure while resolving the feasibility of therapeutically targeting myo7aa −/− mutant ribbons. In this report, we evaluated ribbon synapse number, volume, surface area, and sphericity. Localization of ribbons and their distance from the nearest innervation were also evaluated. We determined that myo7aa −/− mutant ribbon synapses are smaller in volume and surface area; however, all other measurements were not significantly different from wild-type zebrafish. Because the ribbon synapses are nearly indistinguishable between the myo7aa −/− mutant and wild type, it suggests that the ribbons are structurally receptive, supporting that therapeutic intervention may be feasible.

The fibrogenic wound-healing response in liver increases stiffness. Here, macro and microheterogeneity of liver stiffness correlate with HSC heterogeneity in a hepatic fibrosis mouse model. Fibrogenic HSCs localized in stiff collagen-high areas upregulate the expression of focal adhesion molecule FHL2, which, in turn, promotes extracellular matrix protein expression. These results demonstrate that stiffness heterogeneity at the whole organ, lobular, and cellular level drives an amplification loop of fibrogenesis through specific focal adhesion molecular pathways.

Abstract Urinary extracellular vesicles (uEVs) have been used as a biomarker tool for detection of low abundance immunoglobulin-based targets in uEVs with heterogeneous high protein mixtures. This protocol outlines a pre-incubation method to minimize non-specific secondary antibody binding. Additionally, we include our approach for standardization of uEV amounts used for sample comparison. We propose that these and knowledge of the detection limit for target proteins are strategies applicable to other laboratories working with complex heterogeneous samples.

Diet-induced insulin resistance (IR) adversely affects human health and life span. We show that muscle-specific overexpression of human mitochondrial transcription factor A (TFAM) attenuates high-fat diet (HFD)–induced fat gain and IR in mice in conjunction with increased energy expenditure and reduced oxidative stress. These TFAM effects on muscle are shown to be exerted by molecular changes that are beyond its direct effect on mitochondrial DNA replication and transcription. TFAM augmented the muscle tricarboxylic acid cycle and citrate synthase facilitating energy expenditure. TFAM enhanced muscle glucose uptake despite increased fatty acid (FA) oxidation in concert with higher β-oxidation capacity to reduce the accumulation of IR-related carnitines and ceramides. TFAM also increased pAMPK expression, explaining enhanced PGC1α and PPARβ, and reversing HFD-induced GLUT4 and pAKT reductions. TFAM-induced mild uncoupling is shown to protect mitochondrial membrane potential against FA-induced uncontrolled depolarization. These coordinated changes conferred protection to TFAM mice against HFD-induced obesity and IR while reducing oxidative stress with potential translational opportunities.

Light chain (AL) amyloidosis is a devastating, complex, and incurable protein misfolding disease.

AbstractImmunoglobulin light chain (AL) amyloidosis is a fatal complication of B‐cell proliferation secondary to deposition of amyloid fibrils in various organs. Urinary exosomes (UEX) are the smallest of the microvesicles excreted in the urine. Previously, we found UEX of patients with AL amyloidosis contained immunoglobulin light chain (LC) oligomers that patients with multiple myeloma did not have. To further explore the role of the LC oligomers, UEX was isolated from an AL amyloidosis patient with progressive renal disease despite achieving a complete response. LC oligomers were identified. Mass spectrometry (MS) of the UEX and serum identified two monoclonal lambda LCs. Proteomics of the trypsin digested amyloid fragments in the kidney by laser microdissection and MS analysis identified a λ6 LC. The cDNA from plasma cell clone was from the IGLV‐ 6‐57 family and it matched the amino acid sequences of the amyloid peptides. The predicted mass of the peptide product of the cDNA matched the mass of one of the two LCs identified in the UEX and serum. UEX combined with MS were able to identify 2 monoclonal lambda LCs that current clinical methods could not. It also identified the amyloidogenic LC which holds potential for response assessment in the future.