Spatiotemporal Characterization of the Cellular and Molecular Contributors to Liver Fibrosis in a Murine Hepatotoxic-Injury Model

Abstract

The interplay between the inflammatory infiltrate and tissue resident cell populations invokes fibrogenesis. However, the temporal and mechanistic contributions of these cells to fibrosis are obscure. To address this issue, liver inflammation, ductular reaction (DR), and fibrosis were induced in C57BL/6 mice by thioacetamide administration for up to 12 weeks. Thioacetamide treatment induced two phases of liver fibrosis. A rapid pericentral inflammatory infiltrate enriched in F4/80(+) monocytes co-localized with SMA(+) myofibroblasts resulted in early collagen deposition, marking the start of an initial fibrotic phase (1 to 6 weeks). An expansion of bone marrow-derived macrophages preceded a second phase, characterized by accelerated progression of fibrosis (>6 weeks) after DR migration from the portal tracts to the centrilobular site of injury, in association with an increase in DR/macrophage interactions. Although chemokine (C-C motif) ligand 2 (CCL2) mRNA was induced rapidly in response to thioacetamide, CCL2 deficiency only partially abrogated fibrosis. In contrast, colony-stimulating factor 1 receptor blockade diminished C-C chemokine receptor type 2 [CCR2(neg) (Ly6C(lo))] monocytes, attenuated the DR, and significantly reduced fibrosis, illustrating the critical role of colony-stimulating factor 1-dependent monocyte/macrophage differentiation and linking the two phases of injury. In response to liver injury, colony-stimulating factor 1 drives early monocyte-mediated myofibroblast activation and collagen deposition, subsequent macrophage differentiation, and their association with the advancing DR, the formation of fibrotic septa, and the progression of liver fibrosis to cirrhosis.

Infiltrating monocytes in liver injury and repair

“…In models of chronic CCl 4 ‐induced liver fibrosis, Ly6C lo monocyte‐derived macrophages were found to be critical for tissue repair, with persisting fibrosis associated with decreased levels of Ly6C lo monocyte‐derived macrophages 37 , 94 . In contrast, a recent study from MacDonald and colleagues 40 using a thioacetamide‐induced chronic fibrosis model showed that blockade of macrophage colony‐stimulating factor receptor (CSF1R, also known as CD115), the receptor for CSF‐1, resulted in decreased Ly6C lo monocyte‐derived macrophages in parallel with decreased fibrosis, suggesting that Ly6C lo monocyte‐derived macrophages are critical for driving fibrosis. Blockade of CSF1R, however, also results in the depletion of resident KCs, and hence the effects of depleted KCs would need to be disentangled before firm conclusions about the causation of reduced fibrosis can be gleaned from this model.…”

“…99 In a mouse model, CSF-1 treatment promoted a limited amount of differentiation of infiltrating F4/80 lo CD11b hi monocytes into F4/80 hi CD11b lo resident macrophages, suggesting that CSF-1/CSF1R may be important in controlling differentiation of monocytes in the liver. 100 In addition, Melino et al 40 show that CSF1R blockade reduced injury in a mouse model of chronic fibrosis in parallel with a reduction in the numbers of Ly6C lo blood monocytes and monocyte-derived macrophages. Finally, the transcription factor Nur77 is critical for the development of Ly6C lo monocytes in the bone marrow and lack of Nur77 results in monocytes with a more proinflammatory phenotype, suggesting that Nur77 may also be important in controlling the phenotypic switch in tissues.…”

“…Following injury, the number of KCs drastically decreases, whereas the number of monocyte‐derived macrophages increases in the liver, although whether or not monocyte‐derived macrophages ultimately replenish the long‐lived KC population has remained an open question 27 , 35 , 36 , 37 , 38 , 39 . Although it has been proposed that infiltrating monocytes do not replenish the decreased KC population, and rather KCs recover through self‐renewal, just this year, three groups have shown that infiltrating bone marrow‐derived monocytes can replenish the KC population 38 , 40 , 41 , 42 . Two of these groups further showed that this replacement by bone marrow‐derived monocytes resulted in self‐renewing macrophages with similar functional and transcriptional profiles to yolk sac‐derived KCs—showing that this is yet an evolving area of research 41 , 42 …”

“…27,[35][36][37][38][39] Although it has been proposed that infiltrating monocytes do not replenish the decreased KC population, and rather KCs recover through self-renewal, just this year, three groups have shown that infiltrating bone marrow-derived monocytes can replenish the KC population. 38,[40][41][42] Two of these groups further showed that this replacement by bone marrow-derived monocytes resulted in self-renewing macrophages with similar functional and transcriptional profiles to yolk sac-derived KCs-showing that this is yet an evolving area of research. 41,42 Although it is clear that KCs, monocyte-derived macrophages and monocytes play key roles in liver injury and the progression to fibrosis, the independent contributions of these populations is more difficult to tease apart because of overlapping phenotypic markers, inflammatory products, mechanisms, and ontogeny.…”

Macrophage polarization and function: new prospects for fibrotic disease

“…They could aggravate liver injury and create a blockade of inflammatory Ly‐6C + IM recruitment using the CCL2‐inhibitor, which might promote HF resolution by inhibiting pro‐inflammatory cytokine production. Furthermore, early recruitment of granulin‐secreting inflammatory monocytes to the liver resulted in a fibrotic microenvironment and promoted pancreatic cancer metastasis 16 . HSC‐derived signaling molecules promote differentiation of liver macrophages with both pro‐inflammatory and pro‐fibrotic functions mediated by IL‐6 and TGF‐β.…”