Rejuvenating bone marrow hematopoietic reserve prevents regeneration failure and hepatic decompensation in animal model of cirrhosis

Abstract

Background and aim: Bone marrow stem cells (BM-SCs) and their progeny play a central role in tissue repair and regeneration. In patients with chronic liver failure, bone marrow (BM) reserve is severally compromised and they showed marked defects in the resolution of injury and infection, leading to liver failure and the onset of decompensation. Whether BM failure is the cause or consequence of liver failure during cirrhosis is not known. In this study, we aimed to determine the underlying relationship between BM failure and regeneration failure in cirrhosis.

Methodology: C57Bl/6(J) mice were used to develop chronic liver injury through intra-peritoneal administration of carbon tetrachloride (CCl4) for 15 weeks (0.1-0.5 ml/kg). Animals were sacrificed to study the transition of cirrhosis and BM defects. To restore the BM-SC reserve; healthy BM cells were infused via intra-BM infusion and assessed for changes in liver injury, regeneration, and BM-SC reserve.

Results: Using a CCl4-induced animal - model of cirrhosis, we showed the loss of BM-SCs reserve occurred before regeneration failure and the onset of non-acute decompensation. Intra-BM infusion of healthy BM cells induced the repopulation of native hematopoietic stem cells (HSCs) in cirrhotic BM. Restoring BM-HSCs reserve augments liver macrophage-mediated clearance of infection and inflammation dampens neutrophil-mediated inflammation, accelerates fibrosis regression, enhances hepatocyte proliferation, and delays the onset of non-acute decompensation.

Conclusion: These findings suggest that loss of BM-HSCs reserve underlies the compromised innate immune function of the liver, drives regeneration failure, and the onset of non-acute decompensation. We further provide the proof-of-concept that rejuvenating BM-HSC reserve can serve as a potential therapeutic approach for preventing regeneration failure and transition to decompensated cirrhosis.

Keywords: chronic liver injury; cirrhosis; hematopoietic stem cells; hepatic decompensation; innate immune response; kupffer cells; regeneration failure.

Figure 1

Loss of BM-HSC reserve precedes regeneration failure and the onset of decompensated cirrhosis. (A) Schematic representation of a progressive mouse model of chronic liver injury. (B) The blood biochemical levels of total bilirubin and ammonia at each time point, (N = 5). (C) The liver sections were stained with Sirius red and quantitate for progressive change in fibrosis between the groups (N = 5). (D) The Representative immunofluorescent staining images of TUNEL+ hepatocytes (GFP+) and its quantitative analysis (20X) (N = 5). (E) The micrographs showing the immunohistochemistry for hepatocyte proliferation (PCNA staining; 20X) at every stage of CCL4 injury with their quantitative analysis (N = 5). (F, G) The F4/80+ kupffer cells were compared for their phagocytic activity post-BMT (N=5). (F) F4/80+ staining for liver kupffer cells is shown by IHC and its quantification (N = 5). (F) Liver kupffer cells were compared for their phagocytosis based on their phagocytic activity at every time of CCL4 injury (N = 5). (H–J) The flow cytometry gating of LSK (LIN-/SCA-1+/c-Kit+) and LSK sub-population based on CD34+/- and Flt3+/- cell surface markers (LT-HSC, ST-HSC, MPPs) (N = 5 to 10). (K) The bar graph representing negative correlation between LT-HSC and MPPs pool during disease progression. (L) The MSC were analyzed based on their stemness to form colonies (CFU-F) per million of BM-MNCs (N = 5). (M) The graph showing the positive correlation between CFU-F and LT-HSC. Arrows marking the key pathways related to innate immune responses, cell cycle and energy metabolism. Images were taken with an EVOS@FL microscope and quantified using ImageJ. Mean ± SEM; *p<0.05, **p<0.01 and ****p<0.0001. (N=40). "ns" stands for non-significant.

Figure 2

Intra-BM infusion of syngeneic healthy BM cells induces repopulation of native LT-HSC. (A) Schematic representation of intra-femoral (IF) infusion of healthy BM cells and points of sacrifice. (B) The bar graph showing the engraftment of the donor (hBMT and cBMT) GFP+ cells in recipient BM cells (N = 8 to 10). (C) The percentage change in LSK population was compared between hBMT, cBMT and controls (vehicle control) mice (N = 5 to 8). (D) The percentage change in LT-HSC at 24H, D11 and D21 post-BMT of recipients’ BM cells, while the green bar showing the donor-derived LT-HSC% in recipients’ BM LT-HSCs (N = 5). (E) The stemness of MSC was compared for CFU-F per million BM-MSCs at 24H, D11 and D21 post-BMT (N = 5 to 8). (F, G) Bubble or dot plot showing the expression of up-regulated and down-regulated pathways of BM-sorted LSK cells from BMT and a control set of mice based on biological processes (BP), cellular components (CC), Reactome pathways (RP), and KEGG pathways; and compared between 24H vs. D11 in both groups (N = 3). Size corresponds to counts, and color shows value. The data were compared based on -1<log2FC>1, P < 0.05 for their significant expression. Mean ± SEM; *p<0.05, **p<0.01 and ****p<0.0001. (N=43). "ns" stands for non-significant.

Figure 3

Restoring BM LT-HSC reserve accelerates regression of fibrosis and regeneration. (A, B) The liver injury was compared based on (A) AST, ALT, and (B) total bilirubin between the control and BMT groups (cBMT and hBMT) at 24H and D11 post-BMT. (C) H&E staining of liver tissue at 24H showed inflammation and necrosis between the control, cBMT and hBMT groups. (D) Sirius red staining showing reduced fibrosis levels from 24H, D11 and D21 between the controls and BMT groups. (E) Hepatocyte proliferation was checked based on PCNA+ staining on liver sections at 24H, D11 and D21 and compared at 20X blindly. Images were taken in an EVOS@FL microscope and quantified using ImageJ. Mean ± SEM; *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. (N=20). "ns" stands for non-significant.

Figure 4

Restoring BM LT-HSC reserve augments innate immune function in cirrhosis. (A, B) The bubble or dot plot showing the expression of up-regulated and down-regulated pathways of BMT and control group proteins isolated from liver tissue; and compared at (A) 24H and (B) D11 post-BMT (N = 5). The data were compared based on -1<log2FC>1, p < 0.05 for their significant expression. (C) Representative IHC stained for F4/80+ cells post-BMT and compared between groups (control and BMT) at 24H and D11. (D) The graph showing the phagocytosis of F4/80+ cells at 24H and D11 and is compared between the groups. Mean ± SEM; *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. (N=45).

Figure 5

Restoring BM LT-HSC reserve ameliorates progression to decompensated cirrhosis. (A) The schematic representation of hBMT during the progression of hepatic decompensation. (B) The graphs showing the biochemical analysis of the SAAG of controls at D21 post-BMT (N = 5). (C) The blood biochemical test was done to compare the levels of AST, ALT, total bilirubin and ammonia post-hBMT. (D) The micrographs representing the fibrosis regression through Sirius red staining (4X) in BMT and control mice, and the graph showing their quantitative analysis at D21 post-BMT. (E, F) The micrograph (20X) showing the (E) TUNEL+ and (F) PCNA+ hepatocytes and their quantitative analysis at D21 post-BMT of treated groups in comparison to controls. (G) The F4/80+ staining done at D21 post-BMT (20X) between the groups. (H) The creatinine levels were compared to test the secondary organ damage. Images were taken in an EVOS@FL microscope and quantified using ImageJ. Mean ± SEM; *p<0.05, **p<0.01. (N=~25).

Figure 6

The concept diagram showing that rejuvenating bone marrow hematopoietic reserve prevents regeneration failure and hepatic decompensation in animal model of cirrhosis. The representative diagram showing progressive loss of BM hematopoietic reserve precedes the liver regeneration failure during the progression of chronic liver injury. Intra-BM infusion rejuvenates BM-stem cell reserve, augments regression of fibrosis, and regeneration, and prevents the onset of decompensated cirrhosis.