Introduction to Liver Immunology and In Vitro Models

Introduction

The liver is a heterogeneous organ that exerts a crucial role in detoxification, metabolism, and production of essential proteins₁. It is characterized by a tolerogenic phenotype due to its connection to the gastrointestinal tract via the portal vein, which delivers a continuous influx of non-self antigens to the hepatic immune environment₂. The tightly regulated interactions between parenchymal and non-parenchymal cells (NPCs) are fundamental to maintain organ homeostasis and prevent excessive inflammation_3,4. Kupffer cells (KCs), the liver-resident macrophages, play a central role in such mechanisms through the production of anti-inflammatory cytokines, such as IL-10. However, once stimulated, they can release a variety of pro-inflammatory cytokines and chemokines (i.e., IL-6, IL-1β, TNF-α, IL-8) and initiate innate immune responses, representing the first line of defense against pathogens_5,6,7,8. In the adult liver, the KC population is composed of resident and monocyte-derived KCs,while the former are associated with tolerogenic functions in homeostasis, the latter are mainly described in inflammatory contexts₉. Besides KCs, hepatocytes and other NPCs, such as liver sinusoidal endothelial cells, can also play a role in mediating inflammatory responses by further activating KCs, recruiting neutrophils and monocytes to the liver, and resolving the inflammation process₁₀. Moreover, both parenchymal cells and NPCs can exert a role as non-classical antigen-presenting cells (APCs), as they express a variety of pattern recognition receptors (PRRs) and class I and II major histocompatibility complex (MHC) molecules that can lead to T-cell activation_{11,12,13,14,15}. However, how these cells interact to regulate innate immune processes is not fully elucidated.

Although animal models provide valuable insights into the mechanisms regulating this intricated crosstalk, they fail to reproduce human disease pathogenesis or predict drug responses₁₆. The failure in depicting such intricate interactions led to severe limitations in the development of new therapies. In the context of gene therapy, the inadequacy of pre-clinical models in predicting innate immune responses in the liver resulted in the development of CD8 + T-cell-mediated cytotoxic responses during clinical trials employing recombinant adeno-associated viruses (rAAVs), ultimately leading to therapy failure_17,18. This is not surprising as cross-species comparisons individuated differences in immune responses to different types of stimuli_16,19,20, highlighting the limitations that these models impose in a pre-clinical setting. Hence, human-based in vitro cell models are needed to unravel the liver cell interactions that regulate innate immune responses.

While cell lines offer easy manipulation and reproducibility, their lack or low expression levels of sensing pathways compared to primary cells hamper their use in innate immune response evaluation_21,22. As such, primary human hepatocytes (PHHs) represent the gold standard for pre-clinical assessment, although difficulties in the maintenance of their function over extended periods have been reported₂₃. However, it is well established that 3D primary hepatic cell cultures exhibit higher long-term viability, metabolic activities, and cell functions compared to 2D cultures_24,25,26. On the other hand, the high costs and the scarce availability of primary materials, especially for the NPC compartment, have limited the development and employment of fully human in vitro 3D pre-clinical models.

Herein, we aimed to develop a primary human cell-based 3D model to characterize the contribution of hepatocytes and KCs to the innate immune response. Our group previously developed a method for culturing PHHs in 3D, in perfusion stirred-tank bioreactors, which allows the maintenance of their phenotype and functionality for extended periods_27,28. We have recently adapted the method to a smaller scale, overcoming limitations imposed by the scarcity of PHHs₂₉. Here, we leveraged such PHH 3D culture to develop a strategy to differentiate KCs from peripheral blood mononuclear cell (PBMC)-derived monocytes, circumventing the limited availability of primary KCs. We hypothesized that by co-culturing PBMC-derived monocytes with PHHs, we would recapitulate KC differentiation, mimicking the KC niche replenishment via recruitment and differentiation of circulating monocytes happening upon liver infection or injury_30,31. Therefore, we developed a protocol to attain efficient differentiation into functional Kupffer-like cells (KLCs), while retaining high PHH viability and identity. The data presented herein show that our primary cells-based PHH:KLC co-culture is suitable for the assessment of innate immune responses within a human-based liver microenvironment, leveraging further studies for a deeper understanding of liver parenchymal and NPC cro