Lack of Kupffer cell depletion in diethylnitrosamine-induced hepatic inflammation

Abstract

Recent evidence published in the Journal of Hepatology shed light on the fate of Kupffer cells (KCs) during hepatic inflammation. The study by Borst et al. elegantly demonstrated that KCs are rapidly depleted and then replaced by monocyte-derived macrophages in the course of viral hepatitis. 1 This report is quite in contrast to the dogma that KCs, as resident tissue macrophages, are self-maintaining cells. Similar observations were reported for hepatic inflammation induced by paracetamol, CCl 4 , or bacterial infection. 2-4 All of these studies show a temporary decline of KC numbers. An editorial in the Journal of Hepatology raised the question ''of whether all liver injuries lead to KC loss". 5 We would, therefore, like to report data on the hepatic immune cell composition in one of the most frequently used animal models in liver cancer studies: diethylnitrosamine (DEN)-induced hepatocellular carcinoma in mice. Although it has been shown before that short-term DEN treatment drives severe liver injury and acute hepatic inflammation, 6 the effects of DEN administration on the composition of myeloid cells are largely unknown. Therefore, we performed a comprehensive flow cytometric analysis upon short-term high-dose DEN treatment to model acute severe liver damage and longterm low-dose DEN treatment to induce hepatocarcinogenesis. The proportion of leukocytes (CD45 + cells) in liver cell suspensions was significantly higher in the DEN-treated animals in the short-term (2.5 ± 0.4-fold in DEN-treated compared to sham-treated livers; p = 0.004), as well as the long-term model (2.10 ± 0.26-fold in DEN-treated compared to sham-treated livers; p = 0.003). The proportion of hepatic macrophages (CD11b + CD11c À NK1.1 À Ly6G À Ly6C lo F4/80 hi) within the leukocyte fraction was significantly decreased in the short-term DEN-treated mice (Fig. 1A). Accordingly, the monocyte/macrophage ratio was highly elevated (19.1 ± 2.0 in DEN-treated mice compared to 2.2 ± 0.4 sham-treated mice; p = 2.39EÀ7). This finding might be interpreted as a KC depletion. However, normalisation of monocyte and macrophage counts to total cell number showed that, while there was indeed an increase in the number of monocytes, no macrophage depletion in the short-term DEN model can be concluded (Fig. 1A). Concordantly, the expression analysis of the C-type lectin domain family 4, member F (Clec4f), which is a specific marker for KCs, revealed no differences between the DEN-and sham-treated animals, neither in the short-term nor in the long-term model (p = 0.62 and p = 0.66, respectively; Fig. 1B). In long-term DEN-treated mice, an increase in both hepatic monocyte and macrophage counts was observed (Fig. 1A). This might be a result of monocytes giving rise to tissue-resident macrophages. Since Clec4f was not elevated in the long-term DEN model, the source and characteristics of the increased proportion of hepatic macrophages, i.e., whether they are derived from infiltrated monocytes and whether they represent a transient state of differentiation towards ...

Unveiling the depletion of Kupffer cells in experimental hepatocarcinogenesis through liver macrophage subtype-specific markers

“…[2][3][4] In response to these findings, and to an Editorial in the Journal of Hepatology 5 questioning whether this pattern is observable in all liver diseases, Kessler et al published their experience in the diethylnitrosamine (DEN) model of hepatocarcinogenesis. 6 They showed that, despite a decrease in the ratio of macrophages over monocytes, the number of hepatic macrophages (defined as CD11b + CD11c À NK1.1 À Ly6G -Ly6C lo F4/80 hi cells) remained stable after short-term exposure to DEN, while both monocytes and macrophages were increased in the liver 22 weeks after DEN injection. The authors interpreted this as a lack of KC depletion in both the acute and chronic liver injury model.…”

“…Furthermore, they pointed to the characterization of macrophages and infiltrating monocytes in experimental hepatocellular carcinoma (HCC) as an outstanding research question. 6 The hepatic macrophage population consists of different subsets, however, which could not be distinguished using the set of markers employed by Kessler et al 6 Depletion of KCs following an acute insult is indeed well-established, 3 while the composition of liver monocytes/macrophages during DEN-induced HCC, and hepatic carcinogenesis in general, is less clear.…”

Altered glucocorticoid metabolism represents a feature of macroph‐aging

“…The ArC amine‐reactive compensation kit (Invitrogen, #A10346), anti‐rat/hamster beads (BD Biosciences, #552845), or anti‐mouse plus beads (BD, #560497) were used to generate compensation controls. All gates were set by using the appropriate fluorescence minus one (FMO) control (Kessler et al., 2019).…”

Reply to: “Unveiling the depletion of Kupffer cells in experimental hepatocarcinogenesis through liver macrophage subtype-specific markers”

“…5 While we and others discovered a rapid KC loss upon infection, 6,7 this phenomenon was initially not detected in diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC). 8 In contrast, KC loss was apparent in 2 other HCC models. 5 These observations led to the question of whether KC loss after liver injury is a general phenomenon, or whether the KC fate is dependent on the nature of the liver insult.…”

“…[12][13][14] While, KC classification under homeostatic conditions is well established, we and others discovered that under inflammatory con-ditions KC classification is more challenging ( Table 1). 4,6,8 Using the recently identified tissue-resident macrophage marker Tim4, Lefere et al followed up on the question of whether the local KC pool is depleted upon acute acetaminophen (APAP)induced liver injury and chronic inflammation. In accordance with previous data, they detected a profound KC loss during both, acute and chronic liver inflammation.…”