S100A6 Activates Kupffer Cells via the p-P38 and p-JNK Pathways to Induce Inflammation, Mononuclear/macrophage Infiltration Sterile Liver Injury in Mice

Abstract

Noninfectious liver injury, including the effects of chemical material, drugs and diet, is a major cause of liver diseases worldwide. In chemical and drugs-induced liver injury, innate inflammatory responses are mediated by extracellular danger signals. The S100 protein can act as danger signals, which can promote the migration and chemotaxis of immune cells, promote the release of various inflammatory cytokines, and regulate the body's inflammatory and immune responses. However, the role of S100A6 in inflammatory response in chemical and drugs-induced sterile liver injury remains unclear. We constructed the model of sterile liver injury induced by carbon tetrachloride (CCl₄)/Paracetamol (APAP) and performed RNA sequencing (RNA-seq) on the liver tissues after injury (days 2 and 5). We analyzed inflammatory protein secretion in the liver tissue supernatant by enzyme-linked immunosorbent assay (ELISA), determined the inflammation response by bioinformatic analysis during sterile liver injury, and assessed mononuclear/macrophage infiltration by immunohistochemistry and flow cytometry. Immunohistochemistry was used to analyze the location of S100A6. We conducted inflammatory factor expression analysis and molecular mechanistic studies in Kupffer cells (KCs) induced by S100A6 using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), ELISA, and western blot in vitro experiments. We performed chemokine CCL2 expression analysis and molecular mechanism studies using the same method. We used a Transwell assay to show the infiltration of mononuclear/macrophage. We here observed that aggravated inflammatory response was shown in CCl₄ and APAP-administrated mice, as evidenced by enhanced production of inflammatory cytokines (TNF-α, IL-1β), and elevated mononuclear/macrophage infiltration and activation of immunity. The expression of S100A6 was significantly increased on day 2 after sterile liver injury, which is primarily produced by injured liver cells. Mechanistic studies established that S100A6 activates Kupffer cells (KCs) via the p-P38, p-JNK and P65 pathways to induce inflammation in vitro. Furthermore, TNF-α can stimulate liver cells via the p-P38 and p-JNK pathways to produce CCL2 and promote the infiltration of mononuclear/macrophage. In summary, we showed that S100A6 plays an important role in regulating inflammation, thus influencing sterile liver injury. Our findings provide novel evidence that S100A6 can as a danger signal that contributes to pro-inflammatory activation through p-P38 and p-JNK pathways in CCl₄ and APAP-induced sterile liver injury in mice. In addition, the inflammatory factor TNF-α induces a large amount of CCL2 production in normal liver cells surrounding the injured area through a paracrine action, which is chemotactic for blood mononuclear/macrophage infiltration.

Keywords: S100A6; inflammation; kupffer cells; mononuclear/macrophage infiltration.