A novel mouse model for stable engraftment of a human immune system and human hepatocytes

Abstract

Hepatic infections by hepatitis B virus (HBV), hepatitis C virus (HCV) and Plasmodium parasites leading to acute or chronic diseases constitute a global health challenge. The species tropism of these hepatotropic pathogens is restricted to chimpanzees and humans, thus model systems to study their pathological mechanisms are severely limited. Although these pathogens infect hepatocytes, disease pathology is intimately related to the degree and quality of the immune response. As a first step to decipher the immune response to infected hepatocytes, we developed an animal model harboring both a human immune system (HIS) and human hepatocytes (HUHEP) in BALB/c Rag2-/- IL-2Rγc-/- NOD.sirpa uPAtg/tg mice. The extent and kinetics of human hepatocyte engraftment were similar between HUHEP and HIS-HUHEP mice. Transplanted human hepatocytes were polarized and mature in vivo, resulting in 20-50% liver chimerism in these models. Human myeloid and lymphoid cell lineages developed at similar frequencies in HIS and HIS-HUHEP mice, and splenic and hepatic compartments were humanized with mature B cells, NK cells and naïve T cells, as well as monocytes and dendritic cells. Taken together, these results demonstrate that HIS-HUHEP mice can be stably (> 5 months) and robustly engrafted with a humanized immune system and chimeric human liver. This novel HIS-HUHEP model provides a platform to investigate human immune responses against hepatotropic pathogens and to test novel drug strategies or vaccine candidates.

Fig 1. Experimental timeframe and analysis of human hepatocyte engraftment in HUHEP and HIS-HUHEP mice.

(A) Time course of the experiment indicating the age of the mice at each step. Human albumin (hAlb) plasma levels were analyzed longitudinally in HUHEP (n = 4) (B) and HIS-HUHEP (n = 5) (C) mice. (D, E, F and G) Immunofluorescence analysis of HUHEP (left) and HIS-HUHEP (right) liver sections for anti-hAlb (red stain) co-stained with either (D) anti-E-Cadherin (green stain), or (E) anti-Occludin (green stain), or (F) anti-human Cyp2C9 (green stain), or (G) anti-human Cyp3A4 (green stain) expression. Nuclei are stained with DAPI (blue). Scale bar represents 100 microm.

Fig 2. Evaluation of circulating human hematopoietic cells in HIS and HIS-HUHEP mice.

Frequency of human leukocytes (hCD45⁺) measured by FACS in the blood of (a) HIS and (B) HIS-HUHEP mice at early (10–13w) and late (19–22w) time points post-hematopoietic stem cell engraftment. Each dot represents a mouse. (C) Relative proportions of T (CD3⁺) and B (CD19⁺) cells within hCD45⁺ cells from the blood of HIS-HUHEP mice sampled longitudinally (n = 10 mice).

Fig 3. Analysis of human immune cells in HIS and HIS-HUHEP mice.

The (A) percentage and (B) total number of hCD45⁺ cells in the bone marrow (BM), thymus, spleen, liver and lymph nodes (LN) in HIS and HIS-HUHEP mice were determined. Absolute numbers of lymphocyte subsets in the (C) spleen and (D) liver of HIS and HIS-HUHEP mice. (E) Total human IgM and IgG concentrations were measured in the plasma of humanized mice. Error bars show standard deviation. (F) Relative proportions of CD4⁺ T_H and CD8⁺ T_C cell subsets within total CD3⁺ T cells in the liver and spleen of HIS and HIS-HUHEP mice. (G) Relative percentages of naive (CD45RA⁺ white bar) or memory (CD45RO⁺ black bar) CD3⁺ T cells in the liver and spleen of HIS and HIS-HUHEP mice. No statistically significant differences were observed between the HIS and HIS-HUHEP groups (unpaired t tests).

Fig 4. Characterization of human myeloid cells in HIS and HIS-HUHEP mice.

Representative FACS contour plots of liver-isolated cells from HIS-HUHEP mice are shown for each cell subset with the parental gating strategy indicated above the plot. Total numbers of human (A) monocytes (hCD45⁺ CD3^- CD14⁺), (B) myeloid dendritic cells (hCD45⁺ CD3^- CD14^- CD11c⁺ HLA-DR⁺), and (C) plasmacytoid dendritic cells (hCD45⁺ CD3^- CD14^- CD123⁺ HLA-DR⁺) were determined in the liver and spleen of HIS and HIS-HUHEP mice. * p< 0,05 unpaired two-tailed t test.

Fig 5. Evaluation of human immune cells and human hepatocytes in HIS-HUHEP mice.

(a) Correlation analysis of the percentage of hCD45⁺ cells and hAlbumin in the blood of HIS-HUHEP mice sampled at 18–21w post-HIS and 10–16w post-HUHEP reconstitution. Each dot represents a mouse; correlation was analyzed with Pearson’s Χ² test. (B and C) Immunofluorescence analysis of intrahepatic leukocyte infiltration in (B) HIS and (C) HIS-HUHEP livers showing hCD45⁺ cells (purple) and hAlbumin⁺ cells (green). PV: portal vein, white arrows indicate hCD45⁺ cells. Nuclei are stained with DAPI (blue). Scale bar represents 100 microm.