Implications of innate immune sexual dimorphism for MASLD pathogenesis and treatment.

Growing evidence suggests that metabolic dysfunction-associated steatotic liver disease (MASLD) is significantly higher in men versus women. Increased prevalence is observed in postmenopausal women, suggesting that age and sex (hormones) influence MASLD development and progression. Molecular data further reveal that sex regulates the innate immune responses with an essential role in MASLD progression. To date, there has been limited focus on the role of innate immune sexual dimorphism in MASLD, and differences between men and women are not considered in the current drug discovery landscape. In this review, we summarize the sex disparities and innate immune sexual dimorphism in MASLD pathogenesis. We further highlight the importance of harnessing sexual dimorphism in identifying therapeutic targets, developing pharmacological therapies, and designing (pre-) clinical studies for the personalized treatment for MASLD.

Phenotypic and spatial heterogeneity of brain myeloid cells after stroke is associated with cell ontogeny, tissue damage, and brain connectivity.

Acute stroke triggers extensive changes to myeloid immune cell populations in the brain that may be targets for limiting brain damage and enhancing repair. Immunomodulatory approaches will be most effective with precise manipulation of discrete myeloid cell phenotypes in time and space. Here, we investigate how stroke alters mononuclear myeloid cell composition and phenotypes at single-cell resolution and key spatial patterns. Our results show that multiple reactive microglial states and monocyte-derived populations contribute to an extensive myeloid cell repertoire in post-stroke brains. We identify important overlaps and distinctions among different cell types/states that involve ontogeny- and spatial-related properties. Notably, brain connectivity with infarcted tissue underpins the pattern of local and remote altered cell accumulation and reactivity. Our discoveries suggest a global but anatomically governed brain myeloid cell response to stroke that comprises diverse phenotypes arising through intrinsic cell ontogeny factors interacting with exposure to spatially organized brain damage and neuro-axonal cues.

An integrated gene-to-outcome multimodal database for metabolic dysfunction-associated steatotic liver disease.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the commonest cause of chronic liver disease worldwide and represents an unmet precision medicine challenge. We established a retrospective national cohort of 940 histologically defined patients (55.4% men, 44.6% women; median body mass index 31.3; 32% with type 2 diabetes) covering the complete MASLD severity spectrum, and created a secure, searchable, open resource (SteatoSITE). In 668 cases and 39 controls, we generated hepatic bulk RNA sequencing data and performed differential gene expression and pathway analysis, including exploration of gender-specific differences. A web-based gene browser was also developed. We integrated histopathological assessments, transcriptomic data and 5.67 million days of time-stamped longitudinal electronic health record data to define disease-stage-specific gene expression signatures, pathogenic hepatic cell subpopulations and master regulator networks associated with adverse outcomes in MASLD. We constructed a 15-gene transcriptional risk score to predict future hepatic decompensation events (area under the receiver operating characteristic curve 0.86, 0.81 and 0.83 for 1-, 3- and 5-year risk, respectively). Additionally, thyroid hormone receptor beta regulon activity was identified as a critical suppressor of disease progression. SteatoSITE supports rational biomarker and drug development and facilitates precision medicine approaches for patients with MASLD.

Immunology of human fibrosis.

Fibrosis, defined by the excess deposition of structural and matricellular proteins in the extracellular space, underlies tissue dysfunction in multiple chronic diseases. Approved antifibrotics have proven modest in efficacy, and the immune compartment remains, for the most part, an untapped therapeutic opportunity. Recent single-cell analyses have interrogated human fibrotic tissues, including immune cells. These studies have revealed a conserved profile of scar-associated macrophages, which localize to the fibrotic niche and interact with mesenchymal cells that produce pathological extracellular matrix. Here we review recent advances in the understanding of the fibrotic microenvironment in human diseases, with a focus on immune cell profiles and functional immune-stromal interactions. We also discuss the key role of the immune system in mediating fibrosis regression and highlight avenues for future study to elucidate potential approaches to targeting inflammatory cells in fibrotic disorders.

Intestinal B cells license metabolic T-cell activation in NASH microbiota/antigen-independently and contribute to fibrosis by IgA-FcR signalling.

BACKGROUND & AIMS: The progression of non-alcoholic steatohepatitis (NASH) to fibrosis and hepatocellular carcinoma (HCC) is aggravated by auto-aggressive T cells. The gut-liver axis contributes to NASH, but the mechanisms involved and the consequences for NASH-induced fibrosis and liver cancer remain unknown. We investigated the role of gastrointestinal B cells in the development of NASH, fibrosis and NASH-induced HCC. METHODS: C57BL/6J wild-type (WT), B cell-deficient and different immunoglobulin-deficient or transgenic mice were fed distinct NASH-inducing diets or standard chow for 6 or 12 months, whereafter NASH, fibrosis, and NASH-induced HCC were assessed and analysed. Specific pathogen-free/germ-free WT and µMT mice (containing B cells only in the gastrointestinal tract) were fed a choline-deficient high-fat diet, and treated with an anti-CD20 antibody, whereafter NASH and fibrosis were assessed. Tissue biopsy samples from patients with simple steatosis, NASH and cirrhosis were analysed to correlate the secretion of immunoglobulins to clinicopathological features. Flow cytometry, immunohistochemistry and single-cell RNA-sequencing analysis were performed in liver and gastrointestinal tissue to characterise immune cells in mice and humans. RESULTS: Activated intestinal B cells were increased in mouse and human NASH samples and licensed metabolic T-cell activation to induce NASH independently of antigen specificity and gut microbiota. Genetic or therapeutic depletion of systemic or gastrointestinal B cells prevented or reverted NASH and liver fibrosis. IgA secretion was necessary for fibrosis induction by activating CD11b+CCR2+F4/80+CD11c-FCGR1+ hepatic myeloid cells through an IgA-FcR signalling axis. Similarly, patients with NASH had increased numbers of activated intestinal B cells; additionally, we observed a positive correlation between IgA levels and activated FcRg+ hepatic myeloid cells, as well the extent of liver fibrosis. CONCLUSIONS: Intestinal B cells and the IgA-FcR signalling axis represent potential therapeutic targets for the treatment of NASH. IMPACT AND IMPLICATIONS: There is currently no effective treatment for non-alcoholic steatohepatitis (NASH), which is associated with a substantial healthcare burden and is a growing risk factor for hepatocellular carcinoma (HCC). We have previously shown that NASH is an auto-aggressive condition aggravated, amongst others, by T cells. Therefore, we hypothesized that B cells might have a role in disease induction and progression. Our present work highlights that B cells have a dual role in NASH pathogenesis, being implicated in the activation of auto-aggressive T cells and the development of fibrosis via activation of monocyte-derived macrophages by secreted immunoglobulins (e.g., IgA). Furthermore, we show that the absence of B cells prevented HCC development. B cell-intrinsic signalling pathways, secreted immunoglobulins, and interactions of B cells with other immune cells are potential targets for combinatorial NASH therapies against inflammation and fibrosis.

Macrophages promote anti-androgen resistance in prostate cancer bone disease.

Metastatic castration-resistant prostate cancer (PC) is the final stage of PC that acquires resistance to androgen deprivation therapies (ADT). Despite progresses in understanding of disease mechanisms, the specific contribution of the metastatic microenvironment to ADT resistance remains largely unknown. The current study identified that the macrophage is the major microenvironmental component of bone-metastatic PC in patients. Using a novel in vivo model, we demonstrated that macrophages were critical for enzalutamide resistance through induction of a wound-healing-like response of ECM-receptor gene expression. Mechanistically, macrophages drove resistance through cytokine activin A that induced fibronectin (FN1)-integrin alpha 5 (ITGA5)-tyrosine kinase Src (SRC) signaling cascade in PC cells. This novel mechanism was strongly supported by bioinformatics analysis of patient transcriptomics datasets. Furthermore, macrophage depletion or SRC inhibition using a novel specific inhibitor significantly inhibited resistant growth. Together, our findings elucidated a novel mechanism of macrophage-induced anti-androgen resistance of metastatic PC and a promising therapeutic approach to treat this deadly disease.

Macrophages as key regulators of liver health and disease.

Macrophages are a heterogeneous population of innate immune cells and key cellular components of the liver. Hepatic macrophages consist of embryologically-derived resident Kupffer cells (KC), recruited monocyte-derived macrophages (MDM) and capsular macrophages. Both the diversity and plasticity of hepatic macrophage subsets explain their different functions in the maintenance of hepatic homeostasis and in injury processes in acute and chronic liver diseases. In this review, we assess the evidence for macrophage involvement in regulating both liver health and injury responses in liver diseases including acute liver injury (ALI), chronic liver disease (CLD) (including liver fibrosis) and hepatocellular carcinoma (HCC). In healthy livers, KC display critical functions such as phagocytosis, danger signal recognition, cytokine release, antigen processing and the ability to orchestrate immune responses and maintain immunological tolerance. However, in most liver diseases there is a striking hepatic MDM expansion, which orchestrate both disease progression and regression. Single-cell approaches have transformed our understanding of liver macrophage heterogeneity, dynamics, and functions in both human samples and preclinical models. We will further discuss the new insights provided by these approaches and how they are enabling high-fidelity work to specifically identify pathogenic macrophage subpopulations. Given the important role of macrophages in regulating injury responses in a broad range of settings, there is now a huge interest in developing new therapeutic strategies aimed at targeting macrophages. Therefore, we also review the current approaches being used to modulate macrophage function in liver diseases and discuss the therapeutic potential of targeting macrophage subpopulations as a novel treatment strategy for patients with liver disorders.

Genome-Wide Association Study of NAFLD Using Electronic Health Records.

Genome-wide association studies (GWAS) have identified several risk loci for nonalcoholic fatty liver disease (NAFLD). Previous studies have largely relied on small sample sizes and have assessed quantitative traits. We performed a case-control GWAS in the UK Biobank using recorded diagnosis of NAFLD based on diagnostic codes recommended in recent consensus guidelines. We performed a GWAS of 4,761 cases of NAFLD and 373,227 healthy controls without evidence of NAFLD. Sensitivity analyses were performed excluding other co-existing hepatic pathology, adjusting for body mass index (BMI) and adjusting for alcohol intake. A total of 9,723,654 variants were assessed by logistic regression adjusted for age, sex, genetic principal components, and genotyping batch. We performed a GWAS meta-analysis using available summary association statistics. Six risk loci were identified (P < 5*10-8 ) (apolipoprotein E [APOE], patatin-like phospholipase domain containing 3 [PNPLA3, transmembrane 6 superfamily member 2 [TM6SF2], glucokinase regulator [GCKR], mitochondrial amidoxime reducing component 1 [MARC1], and tribbles pseudokinase 1 [TRIB1]). All loci retained significance in sensitivity analyses without co-existent hepatic pathology and after adjustment for BMI. PNPLA3 and TM6SF2 remained significant after adjustment for alcohol (alcohol intake was known in only 158,388 individuals), with others demonstrating consistent direction and magnitude of effect. All six loci were significant on meta-analysis. Rs429358 (P = 2.17*10-11 ) is a missense variant within the APOE gene determining ϵ4 versus ϵ2/ϵ3 alleles. The ϵ4 allele of APOE offered protection against NAFLD (odds ratio for heterozygotes 0.84 [95% confidence interval 0.78-0.90] and homozygotes 0.64 [0.50-0.79]). Conclusion: This GWAS replicates six known NAFLD-susceptibility loci and confirms that the ϵ4 allele of APOE is associated with protection against NAFLD. The results are consistent with published GWAS using histological and radiological measures of NAFLD, confirming that NAFLD identified through diagnostic codes from consensus guidelines is a valid alternative to more invasive and costly approaches.

Deciphering Mesenchymal Drivers of Human Dupuytren's Disease at Single-Cell Level.

Dupuytren's disease (DD) is a common, progressive fibroproliferative disease affecting the palmar fascia of the hands, causing fingers to irreversibly flex toward the palm with significant loss of function. Surgical treatments are limited; therefore, effective new therapies for DD are urgently required. To identify the key cellular and molecular pathways driving DD, we employed single-cell RNA sequencing, profiling the transcriptomes of 35,250 human single cells from DD, nonpathogenic fascia, and healthy dermis. We identify a DD-specific population of pathogenic PDPN+/FAP+ mesenchymal cells displaying an elevated expression of fibrillar collagens and profibrogenic genes. In silico trajectory analysis reveals resident fibroblasts to be the source of this pathogenic population. To resolve the processes governing DD progression, genes differentially expressed during fibroblast differentiation were identified, including upregulated TNFRSF12A and transcription factor SCX. Knockdown of SCX and blockade of TNFRSF12A inhibited the proliferation and altered the profibrotic gene expression of cultured human FAP+ mesenchymal cells, demonstrating a functional role for these genes in DD. The power of single-cell RNA sequencing is utilized to identify the major pathogenic mesenchymal subpopulations driving DD and the key molecular pathways regulating the DD-specific myofibroblast phenotype. Using this precision medicine approach, inhibition of TNFRSF12A has shown potential clinical utility in the treatment of DD.

Genome-wide analysis identifies gallstone-susceptibility loci including genes regulating gastrointestinal motility.

BACKGROUND AND AIMS: Genome-wide association studies (GWAS) have identified several risk loci for gallstone disease. As with most polygenic traits, it is likely that many genetic determinants are undiscovered. The aim of this study was to identify genetic variants that represent new targets for gallstone research and treatment. APPROACH AND RESULTS: We performed a GWAS of 28,627 gallstone cases and 348,373 controls in the UK Biobank, replicated findings in a Scottish cohort (1089 cases, 5228 controls), and conducted a GWA meta-analysis (43,639 cases, 506,798 controls) with the FinnGen cohort. We assessed pathway enrichment using gene-based then gene-set analysis and tissue expression of identified genes in Genotype-Tissue Expression project data. We constructed a polygenic risk score (PRS) and evaluated phenotypic traits associated with the score. Seventy-five risk loci were identified (p < 5 × 10-8 ), of which 46 were new. Pathway enrichment revealed associations with lipid homeostasis, glucuronidation, phospholipid metabolism, and gastrointestinal motility. Anoctamin 1 (ANO1) and transmembrane Protein 147 (TMEM147), both in novel, replicated loci, are expressed in the gallbladder and gastrointestinal tract. Both regulate gastrointestinal motility. The gallstone risk allele rs7599-A leads to suppression of hepatic TMEM147 expression, suggesting that the protein protects against gallstone formation. The highest decile of the PRS demonstrated a 6-fold increased odds of gallstones compared with the lowest decile. The PRS was strongly associated with increased body mass index, serum liver enzymes, and C-reactive protein concentrations, and decreased lipoprotein cholesterol concentrations. CONCLUSIONS: This GWAS demonstrates the polygenic nature of gallstone risk and identifies 46 novel susceptibility loci. We implicate genes influencing gastrointestinal motility in the pathogenesis of gallstones.

CRIg on liver macrophages clears pathobionts and protects against alcoholic liver disease.

Complement receptor of immunoglobulin superfamily (CRIg) is expressed on liver macrophages and directly binds complement component C3b or Gram-positive bacteria to mediate phagocytosis. CRIg plays important roles in several immune-mediated diseases, but it is not clear how its pathogen recognition and phagocytic functions maintain homeostasis and prevent disease. We previously associated cytolysin-positive Enterococcus faecalis with severity of alcohol-related liver disease. Here, we demonstrate that CRIg is reduced in liver tissues from patients with alcohol-related liver disease. CRIg-deficient mice developed more severe ethanol-induced liver disease than wild-type mice; disease severity was reduced with loss of toll-like receptor 2. CRIg-deficient mice were less efficient than wild-type mice at clearing Gram-positive bacteria such as Enterococcus faecalis that had translocated from gut to liver. Administration of the soluble extracellular domain CRIg-Ig protein protected mice from ethanol-induced steatohepatitis. Our findings indicate that ethanol impairs hepatic clearance of translocated pathobionts, via decreased hepatic CRIg, which facilitates progression of liver disease.

Single-cell RNA-seq reveals CD16- monocytes as key regulators of human monocyte transcriptional response to Toxoplasma.

Monocytes are among the major myeloid cells that respond to Toxoplasma, a ubiquitous foodborne that infects ≥ 1 billion people worldwide, in human peripheral blood. As such, a molecular understanding of human monocyte-Toxoplasma interactions can expedite the development of novel human toxoplasmosis control strategies. Current molecular studies on monocyte-Toxoplasma interactions are based on average cell or parasite responses across bulk cell populations. Although informative, population-level averages of monocyte responses to Toxoplasma have sometimes produced contradictory results, such as whether CCL2 or IL12 define effective monocyte responses to the parasite. Here, we used single-cell dual RNA sequencing (scDual-Seq) to comprehensively define, for the first time, the monocyte and parasite transcriptional responses that underpin human monocyte-Toxoplasma encounters at the single cell level. We report extreme transcriptional variability between individual monocytes. Furthermore, we report that Toxoplasma-exposed and unexposed monocytes are transcriptionally distinguished by a reactive subset of CD14+CD16- monocytes. Functional cytokine assays on sorted monocyte populations show that the infection-distinguishing monocytes secrete high levels of chemokines, such as CCL2 and CXCL5. These findings uncover the Toxoplasma-induced monocyte transcriptional heterogeneity and shed new light on the cell populations that largely define cytokine and chemokine secretion in human monocytes exposed to Toxoplasma.

Kidney Single-Cell Atlas Reveals Myeloid Heterogeneity in Progression and Regression of Kidney Disease.

BACKGROUND: Little is known about the roles of myeloid cell subsets in kidney injury and in the limited ability of the organ to repair itself. Characterizing these cells based only on surface markers using flow cytometry might not provide a full phenotypic picture. Defining these cells at the single-cell, transcriptomic level could reveal myeloid heterogeneity in the progression and regression of kidney disease. METHODS: Integrated droplet- and plate-based single-cell RNA sequencing were used in the murine, reversible, unilateral ureteric obstruction model to dissect the transcriptomic landscape at the single-cell level during renal injury and the resolution of fibrosis. Paired blood exchange tracked the fate of monocytes recruited to the injured kidney. RESULTS: A single-cell atlas of the kidney generated using transcriptomics revealed marked changes in the proportion and gene expression of renal cell types during injury and repair. Conventional flow cytometry markers would not have identified the 12 myeloid cell subsets. Monocytes recruited to the kidney early after injury rapidly adopt a proinflammatory, profibrotic phenotype that expresses Arg1, before transitioning to become Ccr2+ macrophages that accumulate in late injury. Conversely, a novel Mmp12+ macrophage subset acts during repair. CONCLUSIONS: Complementary technologies identified novel myeloid subtypes, based on transcriptomics in single cells, that represent therapeutic targets to inhibit progression or promote regression of kidney disease.

Single-cell analyses and machine learning define hematopoietic progenitor and HSC-like cells derived from human PSCs.

Hematopoietic stem and progenitor cells (HSPCs) develop in distinct waves at various anatomical sites during embryonic development. The in vitro differentiation of human pluripotent stem cells (hPSCs) recapitulates some of these processes; however, it has proven difficult to generate functional hematopoietic stem cells (HSCs). To define the dynamics and heterogeneity of HSPCs that can be generated in vitro from hPSCs, we explored single-cell RNA sequencing (scRNAseq) in combination with single-cell protein expression analysis. Bioinformatics analyses and functional validation defined the transcriptomes of naïve progenitors and erythroid-, megakaryocyte-, and leukocyte-committed progenitors, and we identified CD44, CD326, ICAM2/CD9, and CD18, respectively, as markers of these progenitors. Using an artificial neural network that we trained on scRNAseq derived from human fetal liver, we identified a wide range of hPSC-derived HSPCs phenotypes, including a small group classified as HSCs. This transient HSC-like population decreased as differentiation proceeded, and was completely missing in the data set that had been generated using cells selected on the basis of CD43 expression. By comparing the single-cell transcriptome of in vitro-generated HSC-like cells with those generated within the fetal liver, we identified transcription factors and molecular pathways that can be explored in the future to improve the in vitro production of HSCs.

Single-cell technologies in hepatology: new insights into liver biology and disease pathogenesis.

Liver disease is a major global health-care problem, affecting an estimated 844 million people worldwide. Despite this substantial burden, therapeutic options for liver disease remain limited, in part owing to a paucity of detailed analyses defining the cellular and molecular mechanisms that drive these conditions in humans. Single-cell transcriptomic technologies are transforming our understanding of cellular diversity and function in health and disease. In this Review, we discuss how these technologies have been applied in hepatology, advancing our understanding of cellular heterogeneity and providing novel insights into fundamental liver biology such as the metabolic zonation of hepatocytes, endothelial cells and hepatic stellate cells, and the cellular mechanisms underpinning liver regeneration. Application of these methodologies is also uncovering critical pathophysiological changes driving disease states such as hepatic fibrosis, where distinct populations of macrophages, endothelial cells and mesenchymal cells reside within a spatially distinct fibrotic niche and interact to promote scar formation. In addition, single-cell approaches are starting to dissect key cellular and molecular functions in liver cancer. In the near future, new techniques such as spatial transcriptomics and multiomic approaches will further deepen our understanding of disease pathogenesis, enabling the identification of novel therapeutic targets for patients across the spectrum of liver diseases.

Single-Cell Transcriptomics Uncovers Zonation of Function in the Mesenchyme during Liver Fibrosis.

Iterative liver injury results in progressive fibrosis disrupting hepatic architecture, regeneration potential, and liver function. Hepatic stellate cells (HSCs) are a major source of pathological matrix during fibrosis and are thought to be a functionally homogeneous population. Here, we use single-cell RNA sequencing to deconvolve the hepatic mesenchyme in healthy and fibrotic mouse liver, revealing spatial zonation of HSCs across the hepatic lobule. Furthermore, we show that HSCs partition into topographically diametric lobule regions, designated portal vein-associated HSCs (PaHSCs) and central vein-associated HSCs (CaHSCs). Importantly we uncover functional zonation, identifying CaHSCs as the dominant pathogenic collagen-producing cells in a mouse model of centrilobular fibrosis. Finally, we identify LPAR1 as a therapeutic target on collagen-producing CaHSCs, demonstrating that blockade of LPAR1 inhibits liver fibrosis in a rodent NASH model. Taken together, our work illustrates the power of single-cell transcriptomics to resolve the key collagen-producing cells driving liver fibrosis with high precision.