Selective depletion of macrophages reveals distinct, opposing roles during liver injury and repair

Abstract

Macrophages perform both injury-inducing and repair-promoting tasks in different models of inflammation, leading to a model of macrophage function in which distinct patterns of activation have been proposed. We investigated macrophage function mechanistically in a reversible model of liver injury in which the injury and recovery phases are distinct. Carbon tetrachloride---induced liver fibrosis revealed scar-associated macrophages that persisted throughout recovery. A transgenic mouse (CD11b-DTR) was generated in which macrophages could be selectively depleted. Macrophage depletion when liver fibrosis was advanced resulted in reduced scarring and fewer myofibroblasts. Macrophage depletion during recovery, by contrast, led to a failure of matrix degradation. These data provide the first clear evidence that functionally distinct subpopulations of macrophages exist in the same tissue and that these macrophages play critical roles in both the injury and recovery phases of inflammatory scarring.

Figure 1

The relationship between SAMs, collagenous bands, and HSCs during CCl₄ injury and recovery in the Sprague-Dawley rat. (A) Sirius red staining (magnification, ×100) of liver for collagenous bands at 4, 6, and 12 weeks of disease, and following 28 days of recovery after 12 weeks of injury. (B) ED-1 macrophages (magnification, ×100) are associated with scar tissue after 4, 6, and 12 weeks of disease, and following 28 days of recovery after 12 weeks of injury. (C) Graph of SAMs and α-SMA–positive HSCs at different stages of recovery from CCl₄-induced fibrosis (4 weeks intoxication). Note the rapid decline in HSCs in the early phase of recovery.

Figure 2

Structure of the CD11b-DTR transgene and ablation specificity. (A) Diagram representing the MacT6 construct. The cDNA for the DTR-eGFP fusion gene is inserted between the human CD11b promoter (coordinates –1,704 to +83 relative to transcription start) and human growth hormone (hGH) sequence that provides splicing and polyadenylation sequences. Small half-arrows indicate location of the nucleotide pair used for RT-PCR transcript detection; right-facing arrow, transcription start point; gray boxes, exons; and open boxes, untranslated regions. RT-PCR analysis performed on BM-derived macrophages for the control F4/80 (B) and transgenic DTR-eGFP (C) transcripts. Results shown for WT mice and CD11b-DTR lines 34 (positive-line example) and 35 (negative-line example). The eGFP-hGH oligonucleotide pair specifically amplified the transgene mRNA; amplification products were not evident in samples from WT mice or when reverse transcriptase (RT) was omitted. (D–G) FACS assessment of peritoneal and spleen populations. (D) In WT mice, injection of 2 doses of DT at 25 ng/g mouse weight does not affect either the small population of CD3⁺ T cells (upper left quadrant) or the larger population of F4/80⁺ macrophages (lower right quadrant) in the peritoneal cavity. (E) In heterozygous CD11b-DTR mice receiving DT, the F4/80⁺ population is eliminated while the CD3⁺ cells remain. Neither the CD3⁺ nor the B220⁺ cells in the spleen were affected by DT injection either in WT (F) or CD11b-DTR mice (G). Wright-Geimsa–stained cytospin preparations of BTG-elicited peritoneal cells after DT^mut (H) or DT (I) injection into CD11b-DTR mice. Normal proportions of elicited peritoneal cells were observed with injection of DT^mut, but the absence of macrophages and preponderance of neutrophils was apparent in mice treated with DT.

Figure 3

The effect of DT treatment on SAMs in CD11b-DTR mouse liver at peak fibrosis and after 7 days of spontaneous recovery. (A–D) Photomicrographs (magnification, ×400) showing (A) macrophages (Mφ) associated with areas of scarring after 12 weeks of CCl₄-induced injury; (B) absence of macrophages in an area of scarring at 12 weeks of CCl₄ injury, following 5 days of macrophage depletion; (C) macrophages associated with resolving fibrotic band, at 7 days of recovery from CCl₄ injury; and (D) absence of macrophages in scar following macrophage depletion during spontaneous recovery. (E and F) Area of macrophage in fibrotic bands (E) at peak fibrosis following treatment with i.v. PBS, i.p. DT or i.v. DT, or (F) after 7 days of spontaneous recovery after treatment with i.v. PBS, i.p. DT, or i.v. DT (**P < 0.01).

Figure 4

Components of the fibrotic response at peak fibrosis following CCl₄ injury in the CD11b-DTR mouse, from control and macrophage-depleted animals. (A) Sirius red staining (magnification, ×200) at 12 weeks after CCl₄ injury with no depletion or macrophage depletion. Note extensive perisinusoidal fibrosis that is lost with macrophage depletion. (B) Extensive α-SMA–positive HSCs (magnification, ×200) in the fibrotic bands are seen with no depletion but not following macrophage depletion. (C) Collagen III staining (magnification, ×100) showing both fibrotic bands and perisinusoidal fibrosis with no depletion and following depletion. (D) Elastin staining (magnification, ×200) with no depletion or macrophage depletion. Macrophage depletion during CCl₄ injury results in (E) a reduction of sirius red staining and (F) a reduction of area of α-SMA–positive HSCs. (G) Collagen III and (H) elastin are reduced in fibrotic matrix following macrophage depletion (*P < 0.05, ** P < 0.01).

Figure 5

Components of the resolving fibrosis after 7 days of spontaneous recovery from CCl₄-induced injury in control and macrophage-depleted CD11b-DTR mice. (A) Sirius red–positive material (magnification, ×100) following 7 days of recovery with no depletion or macrophage depletion. Note extensive loss of fibrotic bands and perisinusoidal fibrosis. (B) α-SMA–positive HSCs (magnification, ×200) in the liver after 7 days of recovery with no depletion or macrophage depletion. (C) Collagen III staining (magnification, ×100) following 7 days of recovery with no depletion or macrophage depletion. Note persistence of perisinusoidal fibrosis (arrowheads). (D) Elastin staining (magnification, ×200) with no depletion or macrophage depletion. Macrophage depletion during recovery results in (E) persistence of the total area of fibrosis detected by sirius red staining but (F) nonsignificant persistence of α-SMA–positive HSCs. Depletion of macrophage during recovery results in persistence of (G) excess collagen III and (H) elastin (*P < 0.05).

Figure 6

Y chromosome–positive SAMs after 12 weeks of liver injury in female BALB/c mice chimeric for male BM (magnification, ×600). F4/80-labeled macrophages (red) are located in the region of a scar. Y chromosome–FITC hybridization (green) can be seen in nuclei labeled with DAPI (blue). Note macrophages with nuclei containing Y-chromosomal DNA (arrowheads) and also macrophages without Y-chromosomal DNA (arrows).

Figure 7

TGF-β expression in the fibrotic bands (magnification, ×400). (A) After 12 weeks of disease in the mouse model, fibrotic bands (denoted by arrowheads) contain numerous cells showing immunofluorescence for TGF-β. In addition, cells with the morphology of hepatocytes also express cytoplasmic TGF-β (arrow). (B) Following 7 days of recovery from injury, the fibrotic bands show no evidence of TGF-β expression. In addition, no hepatocytes show TGF-β expression.