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. 2016 Oct;65(4):758-768.
doi: 10.1016/j.jhep.2016.05.037. Epub 2016 Jun 1.

Bone Marrow-Derived and Resident Liver Macrophages Display Unique Transcriptomic Signatures but Similar Biological Functions

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Free PMC article

Bone Marrow-Derived and Resident Liver Macrophages Display Unique Transcriptomic Signatures but Similar Biological Functions

Lynette Beattie et al. J Hepatol. .
Free PMC article

Abstract

Background & aims: Kupffer cells (KCs), the resident tissue macrophages of the liver, play a crucial role in the clearance of pathogens and other particulate materials that reach the systemic circulation. Recent studies have identified KCs as a yolk sac-derived resident macrophage population that is replenished independently of monocytes in the steady state. Although it is now established that following local tissue injury, bone marrow derived monocytes may infiltrate the tissue and differentiate into macrophages, the extent to which newly differentiated macrophages functionally resemble the KCs they have replaced has not been extensively studied.

Methods: We studied the two populations of KCs using intravital microscopy, morphometric analysis and gene expression profiling. An ion homeostasis gene signature, including genes associated with scavenger receptor function and extracellular matrix deposition, allowed discrimination between these two KC sub-types.

Results: Bone marrow derived "KCs" accumulating as a result of genotoxic injury, resemble but are not identical to their yolk sac counterparts. Reflecting the differential expression of scavenger receptors, yolk sac-derived KCs were more effective at accumulating acetylated low density lipoprotein, whereas surprisingly, they were poorer than bone marrow-derived KCs when assessed for uptake of a range of bacterial pathogens. The two KC populations were almost indistinguishable in regard to i) response to lipopolysaccharide challenge, ii) phagocytosis of effete red blood cells and iii) their ability to contain infection and direct granuloma formation against Leishmania donovani, a KC-tropic intracellular parasite.

Conclusions: Bone marrow-derived KCs differentiate locally to resemble yolk sac-derived KC in most but not all respects, with implications for models of infectious diseases, liver injury and bone marrow transplantation. In addition, the gene signature we describe adds to the tools available for distinguishing KC subpopulations based on their ontology.

Lay summary: Liver macrophages play a major role in the control of infections in the liver and in the pathology associated with chronic liver diseases. It was recently shown that liver macrophages can have two different origins, however, the extent to which these populations are functionally distinct remains to be fully addressed. Our study demonstrates that whilst liver macrophages share many features in common, regardless of their origin, some subtle differences in function exist.

Data repository: Gene expression data are available from the European Bioinformatics Institute ArrayExpress data repository (accession number E-MTAB-4954).

Keywords: Kupffer cells; Leishmania; Listeria; Liver macrophages; Neisseria; Salmonella; Scavenger receptors.

Figures

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Fig. 1
Fig. 1
Characterisation of YS- and BM-derived KCs. (A) Experimental approach used to generate irradiation chimeras with GFP+ YS-derived KCs. (B) ALT and (C) AST levels in the serum of mice at different time points post-irradiation as compared to control (non-irradiated) animals. (D) H&E stained sections from the liver of control non-irradiated mice or mice (E) 24 h (F) 3 days or (G) 7 days post-irradiation demonstrating very little liver damage or inflammation as a result of irradiation. (H) Immunofluorescent image demonstrating the presence of GFP+ F4/80+ liver resident KCs and GFP- F4/80+ BM-derived KC in the livers of the chimeras generated in (A) (left) or reciprocal chimeras (right), 6 weeks post-irradiation. GFP (green), F4/80 (red). (I) Flow cytometry analysis of the livers of chimeras generated via the method shown in (A) gated on forward scatter (FSC) and side scatter (SSC), CRIg and F4/80 expression and GFP. (J) Analysis of the percentage of CRIg+ F4/80+ cells that express GFP (left) and the number of CRIg+ F4/80+ GFP+ cells in the liver (right) over time. Symbols represent individual mice and are representative of 2 experiments with 5-6 mice per group. (I) The volume and surface area of KCs in 3 dimensions. (L) 2-photon intravital imaging of YS-derived KCs in the livers of chimeras generated via the method shown in (A) (top) or the reciprocal chimera (bottom). Data were analysed with a non-parametric t test. ∗p <0.05.
Fig. 2
Fig. 2
Transcriptional analysis of YS- and BM-derived KCs. (A) Isolation of YS-derived and BM-derived KCs by high speed fluorescence activated cell sorting according to forward and side scatter, GFP and CRIg. Post-sort purity of GFP+ and GFP- KCs. (B) Giemsa stained cytospins of sorted BM-derived (left) or liver resident (right) KCs. (C) Heat maps demonstrating the differential binding to probe sets across the biological replicates for selected groups of genes. (D) Accumulation of mRNA for selected genes expressed as relative expression to hypoxanthine-guanine phosphoribosyltransferase (HPRT). Individual symbols are representative of KCs sorted from individual mice. Data were analysed using a non-parametric t test. ∗∗p <0.01. (E) Immunofluorescent images demonstrating the expression of MARCO (white) on GFP+ (green) F4/80+ (red) liver resident KCs at 200x (top) and 630x (bottom) magnification.
Fig. 3
Fig. 3
YS and BM-derived KCs have similar capacity to clear neuraminidase treated labelled red blood cells. C57BL/6 recipient mice received B6.mTmG.LysMCre bone marrow and PKH26 labelled red blood cells (A) two weeks or (B) 6 weeks post-irradiation. Hepatic mononuclear cells were prepared and plots gated on CRIg+ and GFP expression to examine four populations. CRIg-GFP+ BM-derived cells. CRIg+GFP+ differentiated BM- derived KCs. CRIg+GFP YS-derived KCs and CRIg-GFP- non-macrophage liver resident cells. The PKH26+ proportion of each population is shown. Data are representative of two independent experiments with at least 4 mice/group. (C) Uptake of acetylated LDL as assessed by flow cytometry within F4/80hi CD11blo GFP+ BM- or GFP- YS-derived KCs expressed as a percentage of each population.
Fig. 4
Fig. 4
LPS responsiveness of YS- and BM-derived KCs. CD45.1/.2 chimeras that were 6 weeks post-irradiation were treated with 100 μg of LPS or sham treated and the KCs isolated 24 h later. Groups of 8 chimeric mice were treated and 2 livers pooled to make 4 individual replicates per treatment group. KCs were sorted into YS- and BM-derived populations based on expression of CD45.1, F4/80, and CRIg. (A) Relative expression of CD163, (B) Marco, (C) Ric3, (D) Timd4 and (E) Clec4f. Data are pooled from 2 separate experiments. Data were tested for normal distribution and then analysed using a one-way ANOVA with post-test. ∗∗∗∗p <0.0001, ∗∗∗p <0.001, ∗∗p <0.01, ∗p <0.05.
Fig. 5
Fig. 5
Uptake of bacterial species by BM- and YS-derived KCs. Two-photon imaging of live liver tissue from B6.MacGreen → CD45.1 chimeras from (A) control mice or mice that were injected with heat-killed, Syto 62 labelled (B) S. typhimurium, (C) Neisseria meningitidis or (D) Listeria monocytogenes. (E) Quantification of bacterial uptake by F4/80hi CD11blo GFP+ BM- or GFP- YS-derived KCs by flow cytometry.
Fig. 6
Fig. 6
Control of Leishmania infection by YS- and BM-derived KCs. (A) Percentage of liver BM-derived (light blue bars) and liver resident (dark blue bars) KCs that are infected at 2 h, 48 h and 7 days post-infection with L. donovani. (B) The number of parasites per cell in BM-derived (light blue bars) and liver resident KCs (dark blue bars). (C) The percentage of inflammatory foci formed at 7 days post-infection that are made up of resident or BM-derived KCs. Data were analysed via Kruskal-Wallis test. ∗p <0.05, ∗∗∗p <0.001. (D) Immunofluorescent images demonstrating an inflammatory focus predominantly made up of liver resident KCs (green). F4/80 (red), L. donovani (white). (E) Immunofluorescent images demonstrating an inflammatory focus predominantly made up of BM-derived KCs (green). F4/80 (red), L. donovani (white). (F) Immunofluorescent images demonstrating an inflammatory focus made up of a mixture of liver resident (green) and BM-derived KCs (red). F4/80 (red), L. donovani (white).

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