Hepatic macrophages but not dendritic cells contribute to liver fibrosis by promoting the survival of activated hepatic stellate cells in mice

Abstract

Although it is well established that hepatic macrophages play a crucial role in the development of liver fibrosis, the underlying mechanisms remain largely elusive. Moreover, it is not known whether other mononuclear phagocytes such as dendritic cells (DCs) contribute to hepatic stellate cell (HSC) activation and liver fibrosis. We show for the first time that hepatic macrophages enhance myofibroblast survival in a nuclear factor kappa B (NF-κB)-dependent manner and thereby promote liver fibrosis. Microarray and pathway analysis revealed no induction of HSC activation pathways by hepatic macrophages but a profound activation of the NF-κB pathway in HSCs. Conversely, depletion of mononuclear phagocytes during fibrogenesis in vivo resulted in suppressed NF-κB activation in HSCs. Macrophage-induced activation of NF-κB in HSCs in vitro and in vivo was mediated by interleukin (IL)-1 and tumor necrosis factor (TNF). Notably, IL-1 and TNF did not promote HSC activation but promoted survival of activated HSCs in vitro and in vivo and thereby increased liver fibrosis, as demonstrated by neutralization in coculture experiments and genetic ablation of IL-1 and TNF receptor in vivo. Coculture and in vivo ablation experiments revealed only a minor contribution to NF-κB activation in HSCs by DCs, and no contribution of DCs to liver fibrosis development, respectively.

Conclusion: Promotion of NF-κB-dependent myofibroblast survival by macrophages but not DCs provides a novel link between inflammation and fibrosis.

Figure 1. Microarray and pathway analysis reveal NF-κB and not fibrogenic activation as the predominant effect of hepatic macrophages on HSCs

A-B. Heatmap from microarrays showing quiescent HSCs, 5 day in vitro-activated HSCs, 5 day in vitro-activated HSCs co-cultured with HM, HSC from mice bile duct-ligated for 15 days and HSCs from mice after 4 CCL₄ injections. The heatmap (left panel) includes all genes that were significantly (adjusted p-value <0.05) and at least 4-fold up-or downregulated in HSCs from BDL livers (A). The heatmap (right panel) includes all genes that were significantly (adjusted p-value <0.05) and at least 4-fold up-or downregulated in HSCs from CCl₄ livers (B). C. Shown is the highest-ranking network from IPA analysis. D. Heatmap of NF-κB-responsive genes with significant (adjusted p-value <0.05) and at least 10-fold induction in response to co-culture with HM.

Figure 2. Hepatic macrophages induce NF-κB but not myofibroblastic activation in HSCs in vitro and in vivo

A-B. Hepatic stellate cells were co-cultured with HM for 5 days (A), followed by determination of NF-κB responsive genes by qPCR, or western blot for αSMA (B). C. HSCs were infected with AdIκBsr or an empty control adenovirus followed by co-culture with HSCs for 24h. mRNA expression of HSC activation markers and NF-κB responsive genes was determined by qPCR. D. HSCs were stimulated with conditioned media from HM for 1h. NF-κB activation was determined by immunofluorescent p65 staining (red fluorescence) in HSCs co-stained for F-actin (green fluorescence). Nuclei were marked by Hoechst (blue fluorescence). E. HSCs were stimulated with conditioned media from HM for various times. NF-κB activation was determined by immunoblot for S536-phosphorylated p65 and IκBα. F. HSCs were transduced with an adenoviral NF-κB reporter. After co-culture with HM for 24h, luciferase activity was determined and is expressed as fold induction compared to HSCs cultured with empty inserts. G. Gene expression patterns were determined in ultrapure unplated HSCs isolated from 15 day bile duct-ligated vehicle-treated mice (n=6 isolations) and clodronate-treated mice (n=4 isolations) mice by a combination of gradient centrifugation and FACS sorting without additional plating. mRNA expression of fibrogenic genes and NF-κB dependent genes was determined by qPCR and expressed in comparison to quiescent HSCs isolated from control wt mice (n=3 isolations). H-I. Hepatic stellate cells were treated with 50 μl of 5mg/ml solution per ml of media or liposomal vehicle for 12h. TNFα- and IL-1β-induced NF-κB reporter activity (H.) and cell death, showing Hoechst stained nuclei (blue), retinoids (green) and propidium iodide positive nuclei (red) were determined (I). **p<0.01 in comparison to HSCs cultured without HM. p<0.05 in comparison to HSCs cultured in the absence of HM ## p<0.01 in comparison to AdShuttle-infected HSCs co-cultured with HM. p<0.05, p<0.01 vs HSC BDL Veh.

Figure 3. Macrophage-derived IL-1 and TNF promote NF-κB activation but not myfibroblastic activation in HSCs

A. NF-κB-driven luciferase activity was determined in HSCs that were co-cultured with HM, or treated with IL-1β (5 ng/ml) or TNFα (30 ng/mL) for 24h. B. NF-κB-driven luciferase activity was determined in HSC co-cultured with HM in the presence of vehicle (0.1%BSA in PBS), antagonistic TNFRI-Fc chimera (TFc, 0.5 μg/ml), antagonistic IL-1RI-Fc chimera (IL-Fc, 0.5 μg/ml) or both for 24h. C. mRNA expression of HSC activation markers or NF-κB responsive genes was determined by qPCR in HSC co-cultured with HM in the presence or absence of TNFRI-Fc chimera, IL-1RI-Fc chimera or both for 24h. D-E. Macrophages isolated from BDL livers were treated with treated 16 hours with LPS (10 ng/mL) and interferon gamma (10 ng/mL) to convert them into M1 phenotype (D), or with IL-4 (10 ng/ml) and IL-10 (10 ng/ml) to convert them into M2 phenotype (E). Following 5 washes, M1 or M2 macrophages were co-cultured with HSCs. Co-culture induced expression of NF-κB-dependent genes was determined by qPCR. F. HSCs were treated with rmIL-1β (5 ng/ml) or rmTNFα (30 ng/ml) for 6h followed by analysis of HSC activation markers and NF-κB dependent genes by PCR. G. αSMA levels were analyzed by immunoblotting after treating HSCs with rmIL-1β or rmTNF for 5 days. *p<0.05, ** p<0.01

Figure 4. Hepatic macrophages protect HSCs from cell death

A. HSCs were starved in 0.1% FBS in the presence or absence of HM, and the absence or presence of antagonistic TNFRI-Fc and IL-1RI-Fc chimera. Cell death was determined by propidium iodide staining (red). Nuclei were visualized by Hoechst (blue). Vitamin A-containing HSC lipid droplets were visualized by autofluorescence (pseudocolored in green). Shown are representative images and a quantification of the average of three independent HSC and HM isolations. Insert shows cleaved caspase 3 immunofluorescent staining (red) and Hoechst (blue). B. To determine apoptosis in collagen-expressing myofibroblasts, collagen-GFP reporter mice were treated with vehicle (n=5) or clodronate (n=4) followed by BDL for 9 days and TUNEL staining of liver sections. Cell death of collagen-expressing myofibroblasts was visualized by confocal microscopy and quantified by counting cells double-positive cells for GFP (green) and TUNEL (red). Nuclei were visualized by Hoechst staining (blue). C. Hepatic levels of IL-1β and TNFα mRNA were determined by qPCR in bile duct-ligated vehicle (n=17) and clodronate-treated (n=10) mice. *p<0.05; **p<0.01.

Figure 5. TNF and IL-1 mediate NF-κB activation and protection from cell death in HSCs during liver fibrosis

A. TNFRI and IL1-RI dko (n=5) and wild-type mice (n=10) underwent BDL for 14 days. Hepatic fibrosis was assessed by Sirius Red staining and polarized microscopy, αSMA immunoblotting and hydroxyproline assay. B. TNFRI and IL1-RI dko (n=7) and wild-type mice (n=9) underwent BDL for 5 days. Hepatic fibrogenesis was assessed qPCR for profibrogenic genes, hepatic injury by ALT measurement. C. Following 2 weeks of BDL, HSCs were isolated from mice TNFRI and IL1-RI dko (n=5 isolations) or wild-type mice (n=5 isolations) by gradient centrifugation and Vitamin A fluorescence-based FACS without plating. mRNA expression of fibrogenic and NF-κB dependent genes was determined by qPCR and expressed in comparison to quiescent HSCs isolated from wild-type mice (n=3 isolations). D. To determine apoptosis in HSCs, liver sections from 14 day bile-duct ligated TNFRI and IL1-RI dko (n=5) and wild-type mice (n=5) were stained for desmin (green fluorescence) and by TUNEL (red fluorescence). Nuclei were marked by Hoechst staining (blue fluorescence). Cells positive for both desmin and TUNEL were quantified. *p<0.05; **p<0.01. E. Heatmap of TNF receptor family members including Trail and Trail decoy receptors under different conditions, determined by microarray analysis (left panel). Expression of Trail decoy receptors Tnfrsf22 and Tnfrsf23 as determined by qPCR in ultrapure unplated HSCs isolated from 14 day bile duct-ligated wild-type mice (n=5 isolations) and double-deficient for TNFRI and IL1-RI mice (“dko”, n=5 isolations) by a combination of gradient centrifugation and FACS analysis without additional plating (center and right panel). Data is expressed as fold induction in comparison to quiescent HSCs isolated from control wt mice (n=3 isolations) or as ratio between the investigated mRNA and 18s mRNA if mRNA expression was not detectable in qHSC. *p<0.05; **p<0.01. nd: non-detectable.

Figure 6. Dendritic cells moderately induce NF-κB dependent gene transcription in HSCs but do not contribute to BDL- and CCl₄-induced liver fibrosis

A. Bile duct ligated mice were injected every 5 days for a total of 3 injections with PBS (n=4) or clodronate (n=3) followed by quantification of cDC using CD11c and MHCII flow cytometry FACS plots show percentage of double-positive cells, the bar graph shows cell numbers per gram liver. B. mRNA expression of HSC activation markers (left panel), NF-κB responsive genes (middle panel) or NF-κB-driven luciferase activity (right panel) were determined by qPCR and NF-κB reporter assay, respectively, in HSC co-cultured with DC in the presence of antagonistic TNFRI-Fc and IL-1RI-Fc chimera (both 0.5 μg/ml) or vehicle (0.1%BSA in PBS) for 24h. C-D. Liver fibrosis was induced by BDL in CD11c-DTR bone marrow-chimeric mice. Chimeric male CD11c-DTR were treated with two injections of PBS (n=5) or diphtheria toxin (25 ng/g body weight) (n=6). Mice were sacrificed after 7 days. Deposition of fibrillar collagen was determined by Sirius Red staining (left upper panel) and morphometric quantification (right upper panel) (C). Expression of profibrogenic genes Acta2, Col1a1 and TIMP1 was determined by qPCR. Fold induction was calculated to sham-operated control (D). E. Chimeric CD11c-DTR mice (n=4 each group) were used for DC depletion (25 ng/g first two weeks, 10 ng/g last two weeks) and simultaneous liver fibrosis induction using CCl₄ (0.5 μl/g, three time per week). Fibrosis was determined by Sirius Red staining. F. For pDC depletion C57Bl/6 mice (n=4) were injected every 48 hours with 120G8 antibody (500 μg/mouse) or isotype control during last two weeks of CCl₄ induced liver fibrosis (0.5 μl/g, three time per week for four weeks). Fibrosis was determined by Sirius Red staining. * p<0.05