Hepatic cholesterol crystals and crown-like structures distinguish NASH from simple steatosis

Abstract

We sought to determine whether hepatic cholesterol crystals are present in patients or mice with nonalcoholic fatty liver disease/nonalcoholic steatohepatitis (NASH), and whether their presence or distribution correlates with the presence of NASH as compared with simple steatosis. We identified, by filipin staining, free cholesterol within hepatocyte lipid droplets in patients with NASH and in C57BL/6J mice that developed NASH following a high-fat high-cholesterol diet. Under polarized light these lipid droplets exhibited strong birefringence suggesting that some of the cholesterol was present in the form of crystals. Activated Kupffer cells aggregated around dead hepatocytes that included strongly birefringent cholesterol crystals, forming "crown-like structures" similar to those recently described in inflamed visceral adipose tissue. These Kupffer cells appeared to process the lipid of dead hepatocytes turning it into activated lipid-laden "foam cells" with numerous small cholesterol-containing droplets. In contrast, hepatocyte lipid droplets in patients and mice with simple steatosis did not exhibit cholesterol crystals and their Kupffer cells did not form crown-like structures or transform into foam cells. Our results suggest that cholesterol crystallization within hepatocyte lipid droplets and aggregation and activation of Kupffer cells in crown-like structures around such droplets represent an important, novel mechanism for progression of simple steatosis to NASH.

Fig. 1.

A, B: Human specimens. Representative liver sections stained with Masson's trichrome from the patients included in this study with simple steatosis (A) or NASH (B). In patients with NASH the sections show moderate steatosis with a lobular inflammatory infiltrate and perisinusoidal (“chicken wire”) fibrosis. In patients with simple steatosis, moderate steatosis is demonstrated with little/no inflammation and no fibrosis (×200 magnification). C–F: Mouse specimens. Representative liver sections stained with Masson's trichrome (C, D) or Sirius red (E, F, viewed with polarized light) from C57BL/6J mice fed either a HF diet or a HFHC diet for 30 weeks. Mice on a HFHC diet (D, F) developed severe steatosis with substantial inflammatory infiltrate and perisinusoidal, chicken wire fibrosis consistent with NASH. Mice on a HF diet (C, E) developed moderate steatosis with little inflammatory infiltrate and no fibrosis (simple steatosis) (×200 magnification).

Fig. 2.

Human (1A–H) and mouse (2A–H) frozen liver sections from livers with either simple steatosis (A–D) or NASH (E–H) (×200 magnification). A, E: Bright field images (no stain). B, F: Sections (A) and (E) viewed with polarized light to show colocalization of lipid droplets with birefringence in NASH. C, G: Filipin stain (blue) for free cholesterol. D, H: Sections (C) and (G) viewed with polarized light to show colocalization of filipin staining and birefringence in NASH. Livers with NASH exhibited strongly birefringent crystals within a large proportion of lipid droplets when viewed under polarized light (F, H). Filipin, which stains free cholesterol, also stained prominently the same droplets that had birefringence in livers with NASH, suggesting that the birefringence was due to cholesterol crystals. In contrast, livers with simple steatosis (A–D) did not exhibit any birefringence under polarized light despite having multiple lipid droplets within hepatocytes as seen in the bright field image (A) and did not contain excessive free cholesterol [absence of blue stain in (C)].

Fig. 3.

Human (A, B) and mouse (C–F) liver sections with NASH stained with anti-CD68 antibody [stains green and identifies Kupffer cells (macrophages) (A, C)] or filipin [(B, D, F) stains blue and identifies unesterified cholesterol] and viewed using a fluorescent microscope (A–D, F) or viewed with polarized light (E) to identify birefringent crystals. Kupffer cells (green) surround hepatocytes and stain very strongly for free cholesterol (blue) as shown by the similarity in the blue and green patterns in (A) and (B) (which are the same human liver section) or (C) and (D) (which are the same mouse liver section). The strongly birefringent crystalline material (E) is shown to colocalize with free cholesterol in (F) by the similarity of the patterns of (E) and (F) which are the same mouse liver section. Taken together these sections suggest that in NASH, Kupffer cells aggregate around hepatocytes that contain cholesterol crystals and accumulate cholesterol (×800 magnification).

Fig. 4.

Liver sections of mice on a HFHC diet (NASH) stained with anti-CD68 [stains green and identifies Kupffer cells (A–D)] or anti-CD11b [stains brown and identifies activated Kupffer cells (E)] or anti-TNFα [stains green and identifies activated Kupffer cells (F)] and viewed with fluorescent microscopy (A, C, F), polarized light (B, D) to identify birefringent crystals, or bright field microscopy (E). Pairs (A, B) and (C, D) are photomicrographs of the same liver section viewed with either fluorescent or polarized light showing Kupffer cells (green) aggregating around intensely birefringent lipid droplets containing cholesterol in crystallized form that creates a Maltese cross appearance. CD11b stain in panel (E) and TNFα staining in panel (F) suggest that the Kupffer cells are activated and show morphology very similar to previously described crown-like structures in inflamed visceral adipose tissue. We confirmed that TNFα colocalizes on CD68-positive cells (Kupffer cells) in supplementary Fig. IV [(A, B, E, F) are ×200 magnification; (C, D) are ×800 magnification].

Fig. 5.

Liver sections of mice on a HFHC diet (NASH) stained with osmium and methylene blue (A–C) or viewed with transmission electron microscopy (D, E). Osmium staining with methylene blue counterstaining readily identifies multiple crown-like structures because the remnant lipid droplet of the dead hepatocyte does not take up osmium (marked with asterisks) while all other normal lipid droplets (LD) in viable hepatocytes take up osmium and stain gray/brown. Higher magnification of two of the crown-like structures in panel (A) is shown in panels (B) and (C), demonstrating a granular precipitate within the remnant lipid droplets (marked with asterisks) that are surrounded by Kupffer cells (white arrows). Panel (C) shows that the Kupffer cells acquire multiple small non-osmium binding cholesterol droplets (black arrow) as processing of the remnant lipid droplet of the dead hepatocyte advances. Crown-like structures viewed by electron microscopy are shown in (D) and (E) again demonstrating multiple Kupffer cells (white arrows) surrounding and processing remnant lipid droplets (marked with asterisks) of dead hepatocytes. These remnant droplets do not take osmium (which is used in electron microscopy fixation) and are shown again to contain a granular precipitate in contrast to healthy hepatocyte lipid droplets (LD) that stain gray. In (E) the Kupffer cells have acquired multiple small cholesterol droplets (black arrows) turning into foam cells. The remnant lipid droplets of dead hepatocytes that do not stain with osmium correspond to the intensely birefringent droplets (Maltese crosses) seen by polarized light in Fig. 4B and D.