Distinct immunometabolic signatures in circulating immune cells define disease outcome in acute-on-chronic liver failure.

BACKGROUND AIMS: Acute-on-chronic liver failure (ACLF) is a complication of cirrhosis characterized by multiple organ failure and high short-term mortality. The pathophysiology of ACLF involves elevated systemic inflammation leading to organ failure, along with immune dysfunction that heightens susceptibility to bacterial infections. However, it is unclear how these aspects are associated with recovery and non-recovery in ACLF. APPROACH RESULTS: Here we mapped the single-cell transcriptome of circulating immune cells from ACLF-, acute decompensated (AD) cirrhosis patients and healthy individuals. We further interrogate how these findings as well as immunometabolic- and functional profiles associate with ACLF recovery (ACLF-R) or non-recovery (ACLF-NR). Our analysis unveiled two distinct states of classical monocytes (cMon). Hereto, ACLF-R cMons were characterized by transcripts associated with immune- and stress tolerance, including anti-inflammatory genes such as RETN and LGALS1 . Additional metabolomic- and functional validation experiments implicated an elevated oxidative phosphorylation metabolic program as well as an impaired ACLF-R cMon functionality. Interestingly, we observed a common stress-induced tolerant state, oxidative phosphorylation program and blunted activation among lymphoid populations in ACLF-R patients. Conversely, ACLF-NR cMon featured elevated expression of inflammatory- and stress response genes such as VIM , LGALS2 , and TREM1 along with blunted metabolic activity and increased functionality. CONCLUSIONS: This study identifies distinct immuno-metabolic cellular states that contribute to disease outcome in ACLF patients. Our findings provide valuable insights into the pathogenesis of ACLF, shedding light on factors driving either recovery or non-recovery phenotypes which may be harnessed as potential therapeutic targets in the future.

Comparison of the single-cell and single-nucleus hepatic myeloid landscape within decompensated cirrhosis patients.

Background and aims: A complete understanding of disease pathophysiology in advanced liver disease is hampered by the challenges posed by clinical specimen collection. Notably, in these patients, a transjugular liver biopsy (TJB) is the only safe way to obtain liver tissue. However, it remains unclear whether successful sequencing of this extremely small and fragile tissue can be achieved for downstream characterization of the hepatic landscape. Methods: Here we leveraged in-house available single-cell RNA-sequencing (scRNA-seq) and single-nucleus (snRNA-seq) technologies and accompanying tissue processing protocols and performed an in-patient comparison on TJB's from decompensated cirrhosis patients (n = 3). Results: We confirmed a high concordance between nuclear and whole cell transcriptomes and captured 31,410 single nuclei and 6,152 single cells, respectively. The two platforms revealed similar diversity since all 8 major cell types could be identified, albeit with different cellular proportions thereof. Most importantly, hepatocytes were most abundant in snRNA-seq, while lymphocyte frequencies were elevated in scRNA-seq. We next focused our attention on hepatic myeloid cells due to their key role in injury and repair during chronic liver disease. Comparison of their transcriptional signatures indicated that these were largely overlapping between the two platforms. However, the scRNA-seq platform failed to recover sufficient Kupffer cell numbers, and other monocytes/macrophages featured elevated expression of stress-related parameters. Conclusion: Our results indicate that single-nucleus transcriptome sequencing provides an effective means to overcome complications associated with clinical specimen collection and could sufficiently profile all major hepatic cell types including all myeloid cell subsets.

Q586B2 is a crucial virulence factor during the early stages of Trypanosoma brucei infection that is conserved amongst trypanosomatids.

Human African trypanosomiasis or sleeping sickness, caused by the protozoan parasite Trypanosoma brucei, is characterized by the manipulation of the host's immune response to ensure parasite invasion and persistence. Uncovering key molecules that support parasite establishment is a prerequisite to interfere with this process. We identified Q586B2 as a T. brucei protein that induces IL-10 in myeloid cells, which promotes parasite infection invasiveness. Q586B2 is expressed during all T. brucei life stages and is conserved in all Trypanosomatidae. Deleting the Q586B2-encoding Tb927.6.4140 gene in T. brucei results in a decreased peak parasitemia and prolonged survival, without affecting parasite fitness in vitro, yet promoting short stumpy differentiation in vivo. Accordingly, neutralization of Q586B2 with newly generated nanobodies could hamper myeloid-derived IL-10 production and reduce parasitemia. In addition, immunization with Q586B2 delays mortality upon a challenge with various trypanosomes, including Trypanosoma cruzi. Collectively, we uncovered a conserved protein playing an important regulatory role in Trypanosomatid infection establishment.

Adipose tissue macrophage dysfunction is associated with a breach of vascular integrity in NASH.

BACKGROUND & AIMS: In non-alcoholic fatty liver disease (NAFLD), monocytes infiltrate visceral adipose tissue promoting local and hepatic inflammation. However, it remains unclear what drives inflammation and how the immune landscape in adipose tissue differs across the NAFLD severity spectrum. We aimed to assess adipose tissue macrophage (ATM) heterogeneity in a NAFLD cohort. METHODS: Visceral adipose tissue macrophages from lean and obese patients, stratified by NAFLD phenotypes, underwent single-cell RNA sequencing. Adipose tissue vascular integrity and breaching was assessed on a protein level via immunohistochemistry and immunofluorescence to determine targets of interest. RESULTS: We discovered multiple ATM populations, including resident vasculature-associated macrophages (ResVAMs) and distinct metabolically active macrophages (MMacs). Using trajectory analysis, we show that ResVAMs and MMacs are replenished by a common transitional macrophage (TransMac) subtype and that, during NASH, MMacs are not effectively replenished by TransMac precursors. We postulate an accessory role for MMacs and ResVAMs in protecting the adipose tissue vascular barrier, since they both interact with endothelial cells and localize around the vasculature. However, across the NAFLD severity spectrum, alterations occur in these subsets that parallel an adipose tissue vasculature breach characterized by albumin extravasation into the perivascular tissue. CONCLUSIONS: NAFLD-related macrophage dysfunction coincides with a loss of adipose tissue vascular integrity, providing a plausible mechanism by which tissue inflammation is perpetuated in adipose tissue and downstream in the liver. IMPACT AND IMPLICATIONS: Our study describes for the first time the myeloid cell landscape in human visceral adipose tissue at single-cell level within a cohort of well-characterized patients with non-alcoholic fatty liver disease. We report unique non-alcoholic steatohepatitis-specific transcriptional changes within metabolically active macrophages (MMacs) and resident vasculature-associated macrophages (ResVAMs) and we demonstrate their spatial location surrounding the vasculature. These dysfunctional transcriptional macrophage states coincided with the loss of adipose tissue vascular integrity, providing a plausible mechanism by which tissue inflammation is perpetuated in adipose tissue and downstream in the liver. Our study provides a theoretical basis for new therapeutic strategies to be directed towards reinstating the endogenous metabolic, homeostatic and cytoprotective functions of ResVAMs and MMacs, including their role in protecting vascular integrity.

Pyruvate and uridine rescue the metabolic profile of OXPHOS dysfunction.

INTRODUCTION: Primary mitochondrial diseases (PMD) are a large, heterogeneous group of genetic disorders affecting mitochondrial function, mostly by disrupting the oxidative phosphorylation (OXPHOS) system. Understanding the cellular metabolic re-wiring occurring in PMD is crucial for the development of novel diagnostic tools and treatments, as PMD are often complex to diagnose and most of them currently have no effective therapy. OBJECTIVES: To characterize the cellular metabolic consequences of OXPHOS dysfunction and based on the metabolic signature, to design new diagnostic and therapeutic strategies. METHODS: In vitro assays were performed in skin-derived fibroblasts obtained from patients with diverse PMD and validated in pharmacological models of OXPHOS dysfunction. Proliferation was assessed using the Incucyte technology. Steady-state glucose and glutamine tracing studies were performed with LC-MS quantification of cellular metabolites. The therapeutic potential of nutritional supplements was evaluated by assessing their effect on proliferation and on the metabolomics profile. Successful therapies were then tested in a in vivo lethal rotenone model in zebrafish. RESULTS: OXPHOS dysfunction has a unique metabolic signature linked to an NAD+/NADH imbalance including depletion of TCA intermediates and aspartate, and increased levels of glycerol-3-phosphate. Supplementation with pyruvate and uridine fully rescues this altered metabolic profile and the subsequent proliferation deficit. Additionally, in zebrafish, the same nutritional treatment increases the survival after rotenone exposure. CONCLUSIONS: Our findings reinforce the importance of the NAD+/NADH imbalance following OXPHOS dysfunction in PMD and open the door to new diagnostic and therapeutic tools for PMD.