Multispectral Imaging Differentiates Unique Macrophage Profiles in Patients with Distinct Chronic Liver Diseases

Intrahepatic macrophages influence the composition of the microenvironment, host immune response to liver injury, and development of fibrosis. Compared to stellate cells, the role of intrahepatic macrophages in the development of fibrosis remains ill defined. Multispectral imaging allows detection of multiple markers in situ in human formalin-fixed, paraffin-embedded tissue. This cutting-edge technology is ideal for analyzing human liver tissues since it allows spectral unmixing of fluorophore signals, subtraction of auto-fluorescence, and preserves architecture and the in vivo hepatic milieu. We analyzed resident Kupffer cells (CD68+), monocyte-derived macrophages (Mac387+), pro-fibrogenic macrophages (CD163+), and co-expression of pro-inflammatory (CD14) and anti-inflammatory (CD16) markers in liver biopsies from patients with hepatitis C virus (HCV) and different stages of fibrosis. Liver biopsies with advanced fibrosis showed increased accumulation of CD163+, MAC387+ and CD68+ macrophages in the portal tracts when compared to those with minimal fibrosis. Imaging software generated t-distributed stochastic neighbor embedding (t-SNE) plots and phenotype matrices that facilitated comparison of macrophage profiles. These included monocyte-derived (CD68+/Mac387+) and pro-fibrotic/anti-inflammatory (CD163+/CD16+) phenotypes. We established that the utility of this platform could be extended to liver biopsies from patients with other chronic liver diseases including nonalcoholic steatohepatitis and autoimmune hepatitis. Each disease exhibited a unique profile after spectral imaging analysis and this platform holds the potential to identify patients predisposed to progressive liver disease based on the macrophage composition. In summary, spectral imaging is a powerful tool that enables analysis of macrophage profiles in different types of chronic liver diseases and has potential to change the manner in which we evaluate liver biopsies leading to more personalized treatment strategies.