Schistosomajaponicum Eggs Induce a Proinflammatory, Anti-Fibrogenic Phenotype in Hepatic Stellate Cells

Abstract

Hepatic fibrosis induced by egg deposition is the most serious pathology associated with chronic schistosomiasis, in which the hepatic stellate cell (HSC) plays a central role. While the effect of Schistosoma mansoni eggs on the fibrogenic phenotype of HSCs has been investigated, studies determining the effect of eggs of S. japonicum on HSCs are lacking. Disease caused by S. japonicum is much more severe than that resulting from S. mansoni infection so it is important to compare the pathologies caused by these two parasites, to determine whether this phenotype is due to the species interacting differently with the mammalian host. Accordingly, we investigated the effect of S. japonicum eggs on the human HSC cell line, LX-2, with and without TGF-β (Transforming Growth Factor beta) co-treatment, so as to determine the impact on genes associated with fibrogenesis, inflammation and matrix re-organisation. Activation status of HSCs was assessed by αSMA (Alpha Smooth Muscle Actin) immunofluorescence, accumulation of Oil Red O-stained lipid droplets and the relative expression of selected genes associated with activation. The fibrogenic phenotype of HSCs was inhibited by the presence of eggs both with or without TGF-β treatment, as evidenced by a lack of αSMA staining and reduced gene expression of αSMA and Col1A1 (Collagen 1A1). Unlike S. mansoni-treated cells, however, expression of the quiescent HSC marker PPAR-γ (Peroxisome Proliferator-Activated Receptor gamma) was not increased, nor was there accumulation of lipid droplets. In contrast, S. japonicum eggs induced the mRNA expression of MMP-9 (Matrix Metalloproteinase 9), CCL2 (Chemokine (C-C motif) Ligand 2) and IL-6 (Interleukin 6) in HSCs indicating that rather than inducing complete HSC quiescence, the eggs induced a proinflammatory phenotype. These results suggest HSCs in close proximity to S. japonicum eggs in the liver may play a role in the proinflammatory regulation of hepatic granuloma formation.

Figure 1. Effect of S. japonicum eggs on the activated HSC fibrogenic phenotype.

A number of genes; A. αSMA, B. Col1A1, C. CTGF and D. PPARγ, were selected and their gene expression determined in LX-2 cells relative to the housekeeping genes YWHAZ and ATP5β. S. japonicum eggs, both in direct contact or separated by inserts from LX-2 cells, significantly inhibited expression of genes associated with fibrosis αSMA (A) and Col1a1 (B) compared with untreated cells. TGF-β treatment of cells resulted in significantly increased expression of αSMA (A), Col1a1 (B) and CTGF (C). The stimulatory effects of TGF-β on LX-2 cells were blocked by the presence of eggs, with the expression levels of these genes being similar to egg treatment alone, except in the case of CTGF (C) where increased expression levels where not inhibited by co-treatment. S. japonicum eggs failed to induce expression of PPARγ (n=3, SEM).

Figure 2. Effect of S. japonicum eggs on the expression of genes associated with matrix re-organisation and inflammation in LX-2 cells.

Expression of genes associated with matrix re-organisation and inflammation; A. MMP-9, B. CCL2, C. IL-6, D. MMP-2 and E. TIMP-1, were measured for gene expression relative to the housekeeping genes, YWHAZ and ATP5b. Eggs of S. japonicum significantly promoted the expression of genes associated with granuloma regulation (MMP-9 (A), CCL2 (B) and IL-6 (C)) compared with untreated cells. TGF-β treatment of cells resulted in significantly increased expression of MMP-2 (D) and MMP-9 (A), at both 24 and 72h, and TIMP-1 (E) at 24h. (n=3, SEM).

Figure 3. Phase contrast imaging of LX-2 cells.

LX-2 cells grown for 72hs (A) under normal cell culture conditions or treated with (B) TGF-β alone display an activated myofibroblast phenotype with a large, flat appearance. Cells, in (C) direct contact with S. japonicum eggs (arrows), (D) separated from the eggs by inserts or (E) co-treated with eggs + TGF-β (E) leads to the cells becoming smaller, slender and more elongated with some exhibiting cellular processes by 72h. Scale bars represent 50 µm.

Figure 4. Immunocytochemistry of αSMA in LX-2 cells.

LX-2 cells grown for 72h under (A) normal cell culture conditions or treated with (B) TGF-β alone display an activated myofibroblast phenotype with cells staining positively for αSMA stress fibres (red). Cells, in (C) direct contact with S. japonicum eggs, (D) separated from the eggs by inserts, or (E) co-treated with eggs + TGF-β leads to the cells losing positive staining for αSMA. Cell nuclei are stained blue by DAPI. Scale bars are 50µm.

Figure 5. Lipid droplet staining of LX-2 cells.

LX-2 cells grown for 72h were stained for lipid droplets by Oil Red O staining. A (A) positive control of LX-2 cells treated with MDI to induce lipid droplet storage, as described in previous experiments [13,15], was stained and observed to stain for lipid droplets (arrows). No evidence of lipid droplet staining was observed in the LX-2 cells cultured in (B) normal cell culture conditions or it the (C) presence of S. japonicum eggs. Scale bars are 50µm.

Figure 6. αSMA staining of murine granuloma induced by S. japonicum.

This image displays a granuloma stained for a marker of activated HSC, alpha-smooth muscle actin (αSMA), in a murine model of schistosomiasis japonica taken from our image library. Positive staining for αSMA (brown) can clearly be observed around the granuloma periphery but not in the immediate vicinity of the eggs (arrows). Our results indicate that HSCs in the immediate vicinity of eggs would have reduced αSMA staining and reduced collagen production due to the antifibrotic influence of the eggs, while HSC in the granuloma periphery would be activated and observed to express αSMA due to the host profibrogenic response in the absence or reduced concentration of egg antigen. Scale Bar is 50µm.