Targeting Hepatic Stellate Cell PD-L1 Alters Liver Inflammation and Fibrosis in CCl4 Liver Injury Mouse Model

Abstract

Background & aims: Programmed death-ligand 1 (PD-L1) on hepatic stellate cells (HSCs) is required for HSC activation and suppressing T and B lymphocytes. We tested whether targeting HSC PD-L1 influenced liver inflammation and fibrosis in a carbon tetrachloride (CCl₄) injury mouse model.

Methods: HSC-specific PD-L1 knockout (PD-L1^HSCKO) mice were created by crossing Cd274 floxed mice to Collagen1A1-Cre mice. CCl₄ was injected into PD-L1^HSCKO and PD-L1^HSCWT mice twice weekly for 6 weeks. Liver fibrosis was assessed by Trichrome and Picrosirius Red staining; HSC activation was determined by immunofluorescence and Western blot for HSC activation markers; liver inflammation was studied by multiplex immunofluorescence and cytokine profiling. Multiomics was leveraged to determine how targeting PD-L1 altered HSC producing collagens and cytokines/chemokines.

Results: Collagen deposition was reduced in CCl₄-injured PD-L1^HSCKO livers compared with CCl₄-injured PD-L1^HSCWT livers; myofibroblast density was lower in CCl₄-injured PD-L1^HSCKO livers compared with CCl₄-injured PD-L1^HSCWT livers. CCl₄-injured PD-L1^HSCKO livers had higher lymphocyte densities (GranzymeB+, CD8a+, CD20+) but lower Kupffer and myeloid cell densities (F4/80+ and CD11b+) compared with CCl₄-injured PD-L1^HSCWT livers. Serum aspartate aminotransferase and alanine aminotransferase, however, were similarly elevated by CCl₄ in both groups. Spatial and bulk-cell transcriptomics revealed a global transcriptomic change of HSCs induced by PD-L1 targeting. A targeted proteomics identified that HSC secretion of a group of cytokines/chemokines, including growth/differentiation factor 15, granulocyte-macrophage colony-stimulating factor, C-X-C motif and C-C motif chemokines, was altered upon PD-L1 targeting, highlighting the role of HSC PD-L1 in HSC/Kupffer and HSC/myeloid cell interactions during HSC activation and fibrosis development.

Conclusions: Targeting HSC PD-L1 altered HSC transcriptome and liver inflammation, and suppressed liver fibrosis, representing a potential therapeutic strategy for liver fibrosis.

Keywords: Gene Set Enrichment Analysis; Glial Fibrillary Acidic Protein (GFAP); Macrophage; Single-cell RNA Sequencing; Transforming Growth Factor Beta.

Graphical abstract

Figure 1

Liver fibrosis induced by CCl₄ was reduced in PD-L1^HSCKOmice compared to PD-L1^WTmice. (A) Left, PDL1F/FCol1A1Cre (PD-L1^HSCKO) and matched PDL1+/+Col1A1Cre mice (control; PD-L1^HSCwt) were subjected to CCl₄ injection (1 μL CCl₄ per gram of body weight) or corn oil injection (vehicle) 2 times per week for 6 weeks. Mouse liver was isolated, weighed, and photographed at the end point. Representative pictures of the mouse liver are shown. Right, liver weight was elevated by CCl₄ injection compared with oil injection in control mice, and this effect was attenuated in PD-L1^HSCKO mice. ∗∗P < .01; ∗∗∗P < .001 by ANOVA, n = 3, 3, 8, and 11 mice. (B) Murine serum was collected at the end point and serum biochemistry revealed that AST and ALT were elevated by CCl₄ injection compared to oil injection, and the changes were comparable in the 2 groups. ∗∗P < .01; ∗∗∗P < .001 by ANOVA, n = 3, 3, 8, and 11 mice. (C and D) Representative pictures of Trichrome staining (C) and Sirius Red staining (D) of murine liver sections are shown on the left. Quantitative data revealed that collagen deposition induced by CCl₄ was suppressed in PD-L1^HSCKO livers compared with PD-L1^HSCwt livers. ∗∗∗P < .001; ∗∗∗∗P < .0001 by t-test, n = 8 and 11 mice. For quantification, 5 to 10 microscopic fields were randomly selected from a liver section for densitometry analysis with the ImageJ software so that the average density of blue or red was obtained for the mouse. Bars, 400 μm.

Figure 2

Targeting HSC PD-L1 by Cre/LoxP suppresses myofibroblastic activation of HSCs in CCl₄-injured mice. (A and B) Representative pictures of IF staining for αSMA (A) and PDGFRα (B) on murine liver sections are shown on the top. Quantitative data below revealed that αSMA and PDGFRα IF densities were lower in PD-L1^HSCKO livers compared with PD-L1^HSCwt livers. For IF quantification, 5 to 10 microscopic fields were randomly selected from a liver section for IF density analysis with the ImageJ software so as to obtain the average IF density for the mouse. ∗∗∗∗P < .0001 by t-test, n = 8 and 11 mice. Bars, 400 μm. (C) WB analysis demonstrated that in control mice, CCl₄ injection enhanced liver expression of collagen 1, αSMA, desmin, PDGFRα, and PD-L1 compared with oil injection, and this effect of CCl₄ was suppressed in PD-L1^HSCKO mice. ∗P < .05; ∗∗P < .01 by t-test, n = 5 and 5. (D) WB detected cre protein expressed in the liver of control mice as the result of CCl₄ injection. ∗∗∗P < .001 by t-test, n = 3 and 3 mice. (E) HSCs isolated from PD-L1^HSCKO and PD-L1^HSCWT mice were cultured for 5 days and collected for αSMA IF. Confocal microscopy detected that activation of PD-L1^HSCKO HSCs was suppressed compared with PD-L1^HSCWT HSCs in vitro. ∗∗P < .01 by t-test, n = 10 and 15 microscopy fields. Data represent 3 independent repeats with similar results. Bar, 200 μm.

Figure 3

Targeting HSC PD-L1 alters the immune cell landscape of CCl₄-injured murine liver. (A) Multiplex IF and imaging was performed on liver sections of mice that received CCl₄ injection. A set of IF pictures for αSMA (purple), pan-cytokeratin (brown), Granzyme B (red), CD8a (orange), CD20 (white), F4/80 (green), CD11b (rose-red) on PD-L1^HSCWT liver is shown in Aa-Ag. Cell nuclei were counterstained by DAPI (blue). An image containing 4 makers is shown in Ah and 2 markers in Ai. Bars, 100 μm. (B) Representative pictures of F4/80 IF (green, Ba) and αSMA IF (purple, Bb) revealing weak αSMA and F4/80 IF signals in CCl₄-injured PD-L1^HSCKO mouse liver. Cell nuclei were counterstained by DAPI (blue). Bars, 100 μm.

Figure 4

Targeting HSC PD-L1 alters the immune cell landscape of CCl₄-injured murine liver. (A–C) Multiplex IF images for CD8a (orange, A), GranzymeB (red, B), and CD20 (white, C) are shown on the left, with quantitative data on the right. Cell nuclei were stained by DAPI (blue). Targeting HSC PD-L1 led to higher densities of CD8a+ cells (A), GranzymeB+ cells (B), and CD20+ cells (C) in CCl₄-injured murine livers. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001 by t-test, n=3 mice each group. Data of 6 individual mouse livers are also shown. Five to 7 AOIs, each containing more than 500 cells, were randomly selected from a mouse liver section for quantifying CD8a+ cells; 5 to 9 AOIs, each containing more than 1000 cells, were randomly selected from a mouse liver section for quantification of GranzymeB+ cells; and 6 to 9 AOIs, each containing more than 400 cells, were randomly selected from a mouse liver section for quantification of CD20+ cells. Bars, 100 μm. (D and E) Representative IF images of F4/80 (green, D) and CD11b (rose-red, E), and quantitative data are shown. Targeting HSC PD-L1 led to lower densities of F4/80+ cells (D) and CD11b+ cells (E) in CCl₄-injured murine livers. ∗P < .05 by t-test, n = 3 mice per group. Data of 6 individual mouse livers are also shown. Six to 9 AOIs, each containing more than 1600 cells, were randomly selected from a mouse liver section for quantification of F4/80+ cells, and 4 to 11 AOIs, each containing more than 350 cells, were randomly selected from a mouse liver section for quantification of CD11b+ cells. Bars, 100 μm.

Figure 5

Single-cell RNA sequencing (scRNA seq) detected Cd274/PD-L1 transcripts from activated-HSCs/myofibroblasts of CCl4-injured murine liver. (A) GSM6507612 dataset from the series GSE212039 (RS045) was downloaded and analyzed with an R toolkit Seurat. Hepatic cells were divided into 10 groups by the software. (B) Left, hepatic fibroblasts were further divided into 3 clusters. Right, expression of example marker genes by the 3 clusters is shown by a DotPlot. High levels of Gfap, Lrat, and Des transcripts mark Q-HSCs whereas high levels of Des, Col1a1, and Acta2 mark A-HSCs. (C) Two UMAPs revealing Cd274/PD-L1 transcripts in A-HSCs, but not in Q-HSCs. (D) GSM5257942 from the series GSE212047 (RS039) was analyzed by the R toolkit Seurat, and the murine Lrat+ fibroblasts were divided into 12 clusters with their expression of example marker genes shown by a dot-plot. (E) A violin plot revealing Cd274/PD-L1 transcripts in the cells of clusters 2, 4, 7 (n= 29, 21, and 17 cells).

Figure 6

Targeting HSC PD-L1 induces a global transcriptomic change in the myofibroblasts of CCl₄-injured murine liver. (A) Upper, CCl₄-injured liver sections were subjected to spatial transcriptomics with the NanoString GeoMx Digital Spatial Profiler. A-HSCs were labeled by desmin IF (purple), and 2 representative AOIs selected for transcriptomic profiling are shown. Lower, a volcano plot showing 6964 downregulated and 457 upregulated transcripts as the result of targeting PD-L1 of HSCs in mice. The horizontal line indicates P < .05 and vertical lines show a fold change of 2. (B) GSEA with M2 pathways revealed the pathways of HSCs that were impacted by PD-L1 targeting based on NES >1 and P < .05. (C) A gene set related to collagen formation impacted by PD-L1 targeting is shown. The enrichment of gene transcripts is shown by an enrichment plot (left), and the levels of the transcripts are shown by a heatmap (right). P < .0001, n = 5 and 4. The bar represents the minimum (blue) to the maximum expression level (red).

Figure 7

Targeting HSC PD-L1 induces a global transcriptomic change in the myofibroblasts of CCl₄-injured murine liver and cultured human HSCs. (A) Three gene sets affected by targeting HSC PD-L1 are shown. The enrichment of transcripts is shown by the enrichment plots (left) and gene transcript levels are shown by heatmaps (right). P < .0001, n = 5 and 4. The bar represents the minimum (blue) to the maximum expression level (red). (B) A heatmap showing the transcripts encoding ECM in control and PD-L1 KO myofibroblasts as detected by spatial transcriptomics. P < .05 by ANOVA, n = 5 and 4. The bar represents the minimum (blue) to the maximum expression level (red). (C) A heatmap revealing the transcripts encoding ECM in control and PD-L1 knockdown human HSCs as detected by RNA sequencing (GSE167173). (D) A heatmap revealing the transcripts encoding receptors related to murine HSC activation in control and PD-L1 KO myofibroblasts as detected by spatial transcriptomics. P < .05 except Met by ANOVA, n = 5 and 4. (E) A heatmap revealing the transcripts encoding receptors related to HSC activation in control and PD-L1 knockdown human HSCs as detected by RNA sequencing (GSE167173).

Figure 8

Targeting HSC PD-L1 alters cytokine/chemokine transcripts of HSCs and the cytokine repertoire of CCl₄-injured murine liver. (A) A heatmap revealing the transcripts encoding a panel of cytokines/chemokines in control and PD-L1 KO myofibroblasts as detected by spatial transcriptomics. P < .05 by ANOVA except Igfbp3 and Igfbp4, n = 5 and 4. (B) A heatmap revealing the transcripts encoding cytokines/chemokines in control and PD-L1 knockdown human HSCs as detected by RNA sequencing (GSE167173). (C) A heatmap revealing the transcripts encoding another panel of cytokines/chemokines in control and PD-L1 knockout myofibroblasts as detected by spatial transcriptomics. P < .05 by ANOVA except Ccl17, Ccl9, and Cxcl1, n = 5 and 4. (D) A heatmap revealing the transcripts encoding another panel of cytokines/chemokines in control and PD-L1 knockdown human HSCs as detected by RNA sequencing (GSE167173). (E) Liver lysates were subjected to cytokine profiling with a Proteome Profiler Mouse XL Cytokine Array kit (ARY028 R & D System). Forty-three prominent cytokines/chemokines detected with their names are shown on the bottom. (F) A bar graph revealing that 11 cytokines/chemokines were affected by targeting HSC PD-L1 in CCl₄-injured livers. ∗P < .05; ∗∗P < .01 by t-test. n = 4 and 4 mice.

Figure 9

Targeting PD-L1 alters cytokine/chemokine secretion of primary human HSCs. (A) CM of serum-starved HSCs were collected for cytokine profiling with a Proteome Profiler human XL Cytokine Array kit (ARY022B R & D System). Thirty-four prominent cytokines/chemokines detected and their names are shown on the bottom. (B) A bar graph revealing that 15 cytokines/chemokines were affected by targeting PD-L1 of HSCs. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001 by t-test. n = 4 and 4. (C) CM of TGFβ1-stimulated HSCs were collected for cytokine profiling; 34 prominent cytokines/chemokines were detected and their names are shown on the bottom. (D) A bar graph revealing that 14 cytokines/chemokines were altered by PD-L1 targeting. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001 by t-test. n = 4 and 4.

Figure 10

Transcriptional and posttranscriptional mechanisms are involved in cytokine/chemokine secretion of PD-L1 knockdown HSCs. (A) Left, the transcripts of 15 cytokines/chemokines identified from unstimulated HSCs were extracted from GSE167173 with their expression levels shown by a heatmap. Right, 3 bar graphs revealing 7 downregulated, 1 upregulated, and 7 unchanged transcripts in serum-starved PD-L1 knockdown HSCs compared with control HSCs. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001 by t-test. n = 3 and 3. (B) Left, 13 transcripts of the 14 cytokines/chemokines identified from TGFβ1-stimulated HSCs were extracted from GSE167173 with their expression shown in a heatmap. Right, 3 bar graphs revealing 3 downregulated, 3 upregulated, and 7 unchanged transcripts in response to PD-L1 targeting in TGFβ1-stimulated human HSCs. ∗P < .05; ∗∗P < .01 by t-test. n = 3 and 3.

Figure 11

HSCs promote migration and proliferation of monocytes/macrophages by secreting cytokines/chemokines. (A) Left, HSCs were transduced with shRNA lentiviruses and knockdown of THBS1 by shRNA lentiviruses was detected by WB. Right, IF revealed that CXCL1, CXCL5, or GM-CSF was effectively knocked down by shRNA lentiviruses in HSCs. Bars, 20 μm. (B) CD14+ human monocytes were subjected to Boyden chamber assay with CM of HSCs as a stimulant. CM of control HSCs promoted the chemotaxis of monocytes compared with basal medium, and this effect of CM was suppressed by knocking down THBS1, CXCL1, or CXCL5 of HSCs. ∗∗∗∗P < .0001 by ANOVA, n=6. Data represent 3 independent repeats with similar results. (C) A protocol inducing monocyte-to-macrophage differentiation by M-CSF in vitro is shown (4-day-incubation with 50 ng/mL M-CSF followed by another 4-day-incubation with 100 ng/mL M-CSF). Bars, 100 μm. (D) Boyden chamber assay was performed for the chemotaxis of monocyte-derived macrophages. Migrated cells were stained by DAPI, and representative pictures are shown on the top. Bar, 100 μm. The CM of control HSCs promoted the chemotaxis of macrophages, and this effect of CM was suppressed by knocking down THBS1, CXCL1, or CXCL5 of HSCs. ∗∗∗P < .001; ∗∗∗∗P < .0001 by ANOVA, n = 6. Data represent 3 independent repeats with similar results. (E) CD14+ human monocytes were subjected to MTS proliferation assay with the CM of HSCs as a stimulant. The CM of control HSCs promoted monocyte proliferation was compared with basal medium, and this effect of CM was suppressed by knocking down GM-CSF of HSCs. ∗∗∗∗P < .0001 by ANOVA, n = 6. Data represent 3 independent repeats with similar results.

Figure 12

Localization of CD8 T cells in CCl₄-injured murine livers.Left, representative CD8a IF pictures showing CD8 T cells associated with the portal tract (arrowheads; portal tract CD8 T cells), and cells not associated with the portal tract (arrows; lobular CD8 T cells). Trichrome staining pictures are shown on the bottom. Right, quantitative data revealing more portal tract CD8 T cells in CCl₄-injured PD-L1^HSCKO livers compared with CCl₄-injured PD-L1^HSCWT livers. ∗P < .05 by t-test, n = 3 and 3 mice. Data of 6 individual mice are also shown. Bars, 100 μm.