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BY-NC-ND 4.0 license Open Access Published by De Gruyter September 20, 2022

The polarizable and reprogrammable identity of Kupffer cells in Nonalcoholic Steatohepatitis

  • Tarik Zahr ORCID logo , Kevin Sun and Li Qiang EMAIL logo
From the journal Medical Review

Abstract

Kupffer cells (KCs) are the resident macrophages of the liver with similar origins to myeloid-derived macrophages. Once differentiated, KCs exhibit distinct cellular machinery capable of longevity and self-renewal, making them a crucial player in promoting effective intrahepatic communication. However, this gets compromised in disease states like Nonalcoholic Steatohepatitis (NASH), where the loss of embryo-derived KCs (EmKCs) is observed. Despite this, other KC-like and KC-derived populations start to form and contribute to a variety of roles in NASH pathogenesis, often adopting a NASH-associated molecular signature. Here we offer a brief overview of recent reports describing KC polarization and reprogramming in the liver. We describe the complexities of KC cellular identity, their proposed ability to reprogram to fibroblast-like and endothelial-like cells, and the potential implications in NASH.

In this perspective, we provide a brief analysis of recent studies reporting Kupffer cell (KC) reprogramming in Nonalcoholic Steatohepatitis (NASH), whether to a KC-like cell or to other cell types. By doing so, we hope to highlight the complexities of KC identity that can ultimately influence NASH outcomes.

Hepatic macrophages in NASH

Resident KCs and monocyte-derived macrophages (MoMFs) constitute most hepatic macrophages, with the former predominating in healthy livers. Despite falling under the same category of cells espousing macrophage identity, macrophages derived from the bone marrow play a multitude of differing roles from mature KCs in the pathogenesis of NASH. For example, the progression of NASH entails a loss of KCs followed by an influx of macrophages. Myeloid cell diversity in NASH is associated with microanatomical niches within the liver. Signals triggered by the progression of NASH sequentially program myeloid enhancers to initiate monocyte recruitment and infiltration to the liver [1]. Moreover, a third subset of hepatic macrophages arises in response to KC loss that does not share molecular identifiers found in both KCs and recruited macrophages. These distinct cells, known as lipid-associated macrophages (LAMs), are predominantly found in regions of high Desmin expression, correlating with regions within the liver that are riddled with fibrosis [2]. Notably, the same can be observed during liver injury and can expand to include scar-associated macrophages (SAMacs), resembling LAMs in NASH.

Origins of KCs

KCs are the resident macrophages of the liver with a similar prenatal origin story to that of myeloid-derived macrophages, only to diverge upon differentiation and maturation [3]. KC development begins in the embryonic yolk sac, where precursor cells differentiate into fetal macrophages. Upon entering the bloodstream, these cells migrate to the fetal liver to complete their differentiation into long-lived KCs capable of self-renewal. The ability of embryo-derived KCs (EmKCs) to sustain hepatic residency is speculated to be due to their reprogrammable identity, one that gets compromised with diseases like NASH [4].

KC polarizability

Determinants of KC fate are not only inferred from the influx of macrophages, but by damaged and dying hepatocytes, via intrahepatic exposure to lipids, extrahepatic signals from the gut and adipose tissue, and the release of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). A subset of KCs known as KC2 cells, notably expressing CD206 and ESAM, are heavily involved in lipid metabolism and contribute to lipid storage, mediated by the fatty acid transporter CD36 [5]. These cells respond to and signal for the uptake of lipids by hepatocytes, but lipid overload in the liver compromises this process and impairs KC renewal. This is met by a replacement of KCs with an influx of circulating Ly6C + monocytes. Eventually, these cells give rise to self-renewing and fully differentiated monocyte-derived KCs (MoKCs) [6]. MoKCs are unable to mimic the KC-derived signals that promote the proper handling of liver triglycerides and instead function similarly to MoMFs, further exacerbating NASH severity. As such, the depletion of KCs in NASH is dealt with by a compensatory influx of macrophages.

On the other hand, the subsets of EmKCs that evade depletion can either offer protection against or contribute to the progression of NASH. KCs express a cluster of transcription factors to respond to metabolic dysfunction and inflammation. Occurring in both Nonalcoholic Fatty Liver Disease (NAFLD) and NASH, KC-specific LXRs, SREBPs, PPARs, and NFkB respond to hepatic lipotoxicity, glucotoxicity, and inflamed, damaged tissue [7]. Moreover, activated KCs can clear apoptotic hepatocytes via efferocytosis, a process endemic to anti-inflammatory macrophages. Even with this favorable attribution of KCs, the disruption in homeostasis with NASH advancement impairs their self-renewing ability, and bone marrow-derived cells begin to constitute the majority of hepatic macrophages [8]. Transcriptomic studies further confirm this shift in immune cell composition, and more specifically, immune signature, where both infiltrating macrophages and resident KCs become polarized toward a classic proinflammatory phenotype [9]. LXRs in KCs are redistributed to NASH-specific enhancers to promote CD9 and TREM2 upregulation, establishing a shift towards favoring LAMs and leaving KCs in the minority [7]. Given the complexity of the hepatic macrophage pool in steatotic and fibrotic livers, total KC depletion is unlikely. The existence of known populations of KCs within the liver, such as KC1 (CD206loESAM) and KC2 (CD206+ESAM+), is observable in healthy and diseased livers, with the distribution of such populations being altered instead [5]. From this, it is possible that the KCs escaping depletion may espouse molecular machinery that separates them from KCs prone to identity loss in NASH.

Reprogramming KCs to fibroblast-like cells

Notwithstanding depletion, NASH-favoring KCs can further activate immune cells within the hepatic microenvironment, as well as hepatocytes and non-parenchymal cells (NPCs) of the liver like liver sinusoidal endothelial cells (LSECs) and hepatic stellate cells (HSCs) [10]. For instance, KCs can be activated to secrete pro-fibrotic cytokines that promote the differentiation of HSCs to myofibroblasts, influencing the formation of collagen deposits, a hallmark for liver fibrosis [10]. Moreover, using lineage tracing experiments, one report explored the phenomenon of KCs presenting as fibroblast-like cells. By exposing isolated KCs and liver tissue slice cultures to stress, KCs lose their identity and are transcriptionally programmed to express genes characteristic to fibroblasts, such as genes encoding collagens (Col1a1, Col1a2, Col3a1), and Acta2 [11]. This transdifferentiating property of KCs has not been reported in NASH, but other macrophage populations have been shown to undergo a phenotype switch into fibrocytes, capable of secreting collagen [12]. In countering liver fibrosis, a recent report pinpointed the KC-rich miR-690 as a promising mediator. With NASH development, a downregulation in miR-690 is observed, followed by a loss of KCs. Treatment with a miR-690 mimic in NASH-inducible mice blunted the fibrogenic programming of HSCs and restored Clec4f+ EmKC distribution [13]. Elucidating the possibility of transdifferentiating KCs in NASH and deciphering an approach to reverse this trans-differentiation, possibly through miR-690, would hold great therapeutic value.

Reprogramming KCs to endothelial-like cells

Interestingly, KCs can also share several functional aspects with endothelial cells. In studies using isolated NPCs that are positive for KC markers, endothelial cell markers like VCAM-1 and VEGF-R1 receptors [14], as well as ESAM and CD206 [5], are also expressed. The high expression of such markers in LSECs regulates the activation of surrounding cells and contributes to the pathogenesis of NAFLD/NASH. This observation adds to the speculation of whether subsets of KCs, like KC2 cells, are in fact a unique KC-identifying cell type primed to harbor endothelial cell markers (such as ESAM) or if they are an LSEC-identifying cell type with KC origins (arising from trans-differentiation) [14]. Contamination of artefacts from cell remnants when studying these populations on a single-cell level alludes to the possibility that KC-like LSECs are not KC-like [15]. Nevertheless, as macrophage and endothelial progenitor cells develop from hematopoietic precursors, the likelihood of KCs with heterogenous identities to share LSEC-like properties is possible.

Challenging KC identity in NASH

To conclude, manifestations of such complex identities further dispute a binary molecular makeup of KCs and instead allow for the presumption of a reprogrammable cell population capable of a myriad of functions (Figure 1). Loss of a healthy EmKC identity is observed in NASH, and whether it occurs through their replacement by infiltrating immune cells, adoption of a NASH molecular signature, or trans-differentiation to other cell types (Table 1) remains to be fully characterized.

Figure 1: 
Kupffer cell heterogeneity in NASH. Representative schematic of the varying Kupffer cell and KC-like identities involved in the pathogenesis of NASH. Clec4f+, F4/80+ KCs are reliable markers of embryo-derived KCs that make up approximately 20% of the cells found in a healthy liver. NASH triggers a shift in the molecular makeup of hepatic macrophages, where the loss of EmKCs is met with an influx of circulating monocytes, differentiating into lipid-associated macrophages, scar-associated macrophages, and monocyte-derived KCs. The remaining EmKCs adopt a NASH-associated molecular signature and can activate hepatic stellate cells, liver sinusoidal endothelial cells, and may possibly be capable of transdifferentiating to fibroblast-like and endothelial-like cells. Illustration created in BioRender.com. EmKC, embryo-derived KC; LAM, lipid-associated macrophage; SAMac, scar-associated macrophage; MoKC, monocyte-derived KC; HSC, hepatic stellate cell; LSEC, liver sinusoidal endothelial cell; EndoKC, endothelial-like KC. NASH, Nonalcoholic Steatohepatitis.
Figure 1:

Kupffer cell heterogeneity in NASH. Representative schematic of the varying Kupffer cell and KC-like identities involved in the pathogenesis of NASH. Clec4f+, F4/80+ KCs are reliable markers of embryo-derived KCs that make up approximately 20% of the cells found in a healthy liver. NASH triggers a shift in the molecular makeup of hepatic macrophages, where the loss of EmKCs is met with an influx of circulating monocytes, differentiating into lipid-associated macrophages, scar-associated macrophages, and monocyte-derived KCs. The remaining EmKCs adopt a NASH-associated molecular signature and can activate hepatic stellate cells, liver sinusoidal endothelial cells, and may possibly be capable of transdifferentiating to fibroblast-like and endothelial-like cells. Illustration created in BioRender.com. EmKC, embryo-derived KC; LAM, lipid-associated macrophage; SAMac, scar-associated macrophage; MoKC, monocyte-derived KC; HSC, hepatic stellate cell; LSEC, liver sinusoidal endothelial cell; EndoKC, endothelial-like KC. NASH, Nonalcoholic Steatohepatitis.

Table 1:

Reports of Kupffer cell reprogramming.

KC reprogramming Experiment Stage References
KCs adopt a NASH-associated molecular signature (CD9+, TREM2+) distinct to that of EmKCs in healthy livers Single-cell RNA sequencing In vivo [7, 9]
KCs exposed to stress change morphology overtime, accompanied by a change in transcriptional makeup to resemble fibroblast-like cells Lineage tracing experiments in isolated NPCs and liver tissue slice cultures In vitro, ex vivo [11]
KCs restore Clec4f expression and proliferative ability upon miR-690 treatment in NASH Immunohistochemistry in NASH livers of mice In vivo [13]
KCs express molecular identifiers commonly found in endothelial cells, such as ESAM, VEGF-R1, VCAM-1, while maintaining identity (Clec4f+) Single-cell transcriptomics and NPC isolation In vivo, in vitro [5, 14]
  1. NASH, Nonalcoholic Steatohepatitis; KC, Kupffer cell; EmKCs, embryo-derived KCs; NPC, non-parenchymal cell.


Corresponding author: Li Qiang, Naomi Berrie Diabetes Center, Columbia University, 1150 St. Nicholas Ave, New York, NY 10032, USA; and Department of Pathology and Cell Biology, Columbia University, New York, NY 10032, USA, E-mail:

  1. Research funding: None declared.

  2. Author contributions: T.Z. and L.Q. conceptualized the perspective. T.Z wrote the original draft of the manuscript. K.S. reviewed and edited the manuscript. L.Q. is the primary overseer of this report. All authors have accepted responsibility for the entire content of this manuscript and approved its submission.

  3. Competing interests: Authors state no conflict of interest.

  4. Ethical approval: Not applicable.

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Received: 2022-07-25
Accepted: 2022-08-29
Published Online: 2022-09-20
Published in Print: 2022-08-26

© 2022 the author(s), published by De Gruyter, Berlin/Boston

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

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