Heterogeneity in intrahepatic macrophage populations and druggable target expression in patients with steatotic liver disease-related fibrosis.

Background & Aims: Clinical trials for reducing fibrosis in steatotic liver disease (SLD) have targeted macrophages with variable results. We evaluated intrahepatic macrophages in patients with SLD to determine if activity scores or fibrosis stages influenced phenotypes and expression of druggable targets, such as CCR2 and galectin-3. Methods: Liver biopsies from controls or patients with minimal or advanced fibrosis were subject to gene expression analysis using nCounter to determine differences in macrophage-related genes (n = 30). To investigate variability among individual patients, we compared additional biopsies by staining them with multiplex antibody panels (CD68/CD14/CD16/CD163/Mac387 or CD163/CCR2/galectin-3/Mac387) followed by spectral imaging and spatial analysis. Algorithms that utilize deep learning/artificial intelligence were applied to create cell cluster plots, phenotype profile maps, and to determine levels of protein expression (n = 34). Results: Several genes known to be pro-fibrotic (e.g. CD206, TREM2, CD163, and ARG1) showed either no significant differences or significantly decreased with advanced fibrosis. Although marked variability in gene expression was observed in individual patients with cirrhosis, several druggable targets and their ligands (e.g. CCR2, CCR5, CCL2, CCL5, and LGALS3) were significantly increased when compared to patients with minimal fibrosis. Antibody panels identified populations that were significantly increased (e.g. Mac387+), decreased (e.g. CD14+), or enriched (e.g. interactions of Mac387) in patients that had progression of disease or advanced fibrosis. Despite heterogeneity in patients with SLD, several macrophage phenotypes and druggable targets showed a positive correlation with increasing NAFLD activity scores and fibrosis stages. Conclusions: Patients with SLD have markedly varied macrophage- and druggable target-related gene and protein expression in their livers. Several patients had relatively high expression, while others were like controls. Overall, patients with more advanced disease had significantly higher expression of CCR2 and galectin-3 at both the gene and protein levels. Impact and implications: Appreciating individual differences within the hepatic microenvironment of patients with SLD may be paramount to developing effective treatments. These results may explain why such a small percentage of patients have responded to macrophage-targeting therapies and provide additional support for precision medicine-guided treatment of chronic liver diseases.

Patients with fibrosis from non-alcoholic steatohepatitis have heterogeneous intrahepatic macrophages and therapeutic targets.

Background and Aims: In clinical trials for reducing fibrosis in NASH patients, therapeutics that target macrophages have had variable results. We evaluated intrahepatic macrophages in patients with non-alcoholic steatohepatitis to determine if fibrosis influenced phenotypes and expression of CCR2 and Galectin-3. Approach & Results: We used nCounter to analyze liver biopsies from well-matched patients with minimal (n=12) or advanced (n=12) fibrosis to determine which macrophage-related genes would be significantly different. Known therapy targets (e.g., CCR2 and Galectin-3) were significantly increased in patients with cirrhosis.However, several genes (e.g., CD68, CD16, and CD14) did not show significant differences, and CD163, a marker of pro-fibrotic macrophages was significantly decreased with cirrhosis. Next, we analyzed patients with minimal (n=6) or advanced fibrosis (n=5) using approaches that preserved hepatic architecture by multiplex-staining with anti-CD68, Mac387, CD163, CD14, and CD16. Spectral data were analyzed using deep learning/artificial intelligence to determine percentages and spatial relationships. This approach showed patients with advanced fibrosis had increased CD68+, CD16+, Mac387+, CD163+, and CD16+CD163+ populations. Interaction of CD68+ and Mac387+ populations was significantly increased in patients with cirrhosis and enrichment of these same phenotypes in individuals with minimal fibrosis correlated with poor outcomes. Evaluation of a final set of patients (n=4) also showed heterogenous expression of CD163, CCR2, Galectin-3, and Mac387, and significant differences were not dependent on fibrosis stage or NAFLD activity. Conclusions: Approaches that leave hepatic architecture intact, like multispectral imaging, may be paramount to developing effective treatments for NASH. In addition, understanding individual differences in patients may be required for optimal responses to macrophage-targeting therapies.

Primary Hepatic Mesothelial Cyst: A Rare Entity to Be Considered in the Differential Diagnosis of Neonatal Cystic Lesions.

We report two cases of primary hepatic mesothelial cysts in neonates previously identified during perinatal imaging. Both neonatal cases were reimaged in the postnatal period, demonstrating the persistence of these cystic hepatic lesions. In both instances, the decision was made to treat with surgical resection and both patients tolerated the surgery well with no significant postoperative complications. Histopathological examination of these lesions discovered a cuboidal lining that was calretinin and WT1 positive and CD31 negative, indicating the diagnosis of a mesothelial cyst of hepatic origin. These cases bring attention to the broad differential diagnosis of congenital primary hepatic cystic lesions, as well as the diagnostic pathway to confirm a primary hepatic mesothelial cyst.

Macrophage infection, activation, and histopathological findings in ebolavirus infection.

Macrophages contribute to Ebola virus disease through their susceptibility to direct infection, their multi-faceted response to ebolaviruses, and their association with pathological findings in tissues throughout the body. Viral attachment and entry factors, as well as the more recently described influence of cell polarization, shape macrophage susceptibility to direct infection. Moreover, the study of Toll-like receptor 4 and the RIG-I-like receptor pathway in the macrophage response to ebolaviruses highlight important immune signaling pathways contributing to the breadth of macrophage responses. Lastly, the deep histopathological catalogue of macrophage involvement across numerous tissues during infection has been enriched by descriptions of tissues involved in sequelae following acute infection, including: the eye, joints, and the nervous system. Building upon this knowledge base, future opportunities include characterization of macrophage phenotypes beneficial or deleterious to survival, delineation of the specific roles macrophages play in pathological lesion development in affected tissues, and the creation of macrophage-specific therapeutics enhancing the beneficial activities and reducing the deleterious contributions of macrophages to the outcome of Ebola virus disease.

Cutting-Edge Platforms for Analysis of Immune Cells in the Hepatic Microenvironment-Focus on Tumor-Associated Macrophages in Hepatocellular Carcinoma.

The role of tumor-associated macrophages (TAMs) in the pathogenesis of hepatocellular carcinoma (HCC) is poorly understood. Most studies rely on platforms that remove intrahepatic macrophages from the microenvironment prior to evaluation. Cell isolation causes activation and phenotypic changes that may not represent their actual biology and function in situ. State-of-the-art methods provides new strategies to study TAMs without losing the context of tissue architecture and spatial relationship with neighboring cells. These technologies, such as multispectral imaging (e.g., Vectra Polaris), mass cytometry by time-of-flight (e.g., Fluidigm CyTOF), cycling of fluorochromes (e.g., Akoya Biosciences CODEX/PhenoCycler-Fusion, Bruker Canopy, Lunaphore Comet, and CyCIF) and digital spatial profiling or transcriptomics (e.g., GeoMx or Visium, Vizgen Merscope) are being utilized to accurately assess the complex cellular network within the tissue microenvironment. In cancer research, these platforms enable characterization of immune cell phenotypes and expression of potential therapeutic targets, such as PDL-1 and CTLA-4. Newer spatial profiling platforms allow for detection of numerous protein targets, in combination with whole transcriptome analysis, in a single liver biopsy tissue section. Macrophages can also be specifically targeted and analyzed, enabling quantification of both protein and gene expression within specific cell phenotypes, including TAMs. This review describes the workflow of each platform, summarizes recent research using these approaches, and explains the advantages and limitations of each.

Comparison of liver biopsies before and after direct-acting antiviral therapy for hepatitis C and correlation with clinical outcome.

Direct-acting antivirals (DAA) have replaced interferon (IFN)-based therapies for hepatitis C virus. In this retrospective clinical study, we examined differences in histopathologic features in paired liver biopsies collected from the same patient before and after DAA and correlated these findings with clinical outcome. Biopsies (n = 19) were evaluated by quantitative imaging analysis to measure steatosis and fibrosis. Most patients had decreased steatosis in their post-treatment, follow-up biopsies. However, one patient had a striking increase in steatosis (from 0.86 to 6.32%) and later developed decompensated cirrhosis and hepatocellular carcinoma (HCC). This patient had a marked increase in fibrosis between biopsies, with a CPA of 6.74 to 32.02. Another patient, who already had bridging fibrosis at the time of her pre-treatment biopsy, developed cholangiocarcinoma after DAA. Even though the overall inflammatory activity in the post-treatment biopsies significantly decreased after treatment, 60% of patients had persistent portal lymphocytic inflammation. In summary, DAAs decreased steatosis and hepatic inflammation in most patients, although some may have persistence of lymphocytic portal inflammation. Patients known to have advanced fibrosis at treatment initiation and who have other risk factors for ongoing liver injury, such as steatosis, should be followed closely for the development of adverse outcomes, such as portal hypertension and primary liver cancers.

Profile of the average organ non-donor: can it be used predictively?

OBJECTIVE: To describe the profile of the average organ non-donor, compare it to that of the average donor, and identify characteristics that predict the likelihood that a given individual will be a non-donor. METHODS: The charts of 397 consenting potential organ donors of LifeLink of Puerto Rico from 2009 through 2011 were reviewed. Data regarding gender, age, BMI, the presence of diabetes, hypertension and/or kidney injury, death from cerebrovascular accident, and smoking were collected. RESULTS: Of the 397 charts reviewed, 283 were from donors, 96 were from non-donors, and 18 were excluded from the analysis. When compared to donors, non-donors were found more frequently to be 60 years old or older, diabetic, hypertensive, or obese; to have suffered from kidney injury, to have smoked and to have died of a cerebrovascular accident. On multivariate analysis, age, diabetes, kidney injury and smoking remained significant. However, after adjusting for age, only smoking and death from cerebrovascular accident remained statistically associated to non-donor status. CONCLUSION: Although being over 60 years old, having smoked and dying from a cerebrovascular accident were characteristics found significantly more frequently in non-donors, these characteristics were also present in some donors. Therefore, a careful evaluation of each potential donor is still mandatory to avoid the loss of transplantable organs.