Identification of pro-inflammatory CD205+ macrophages in livers of hepatitis B virus transgenic mice and patients with chronic hepatitis B

Abstract

Hepatic macrophages play a central role in disease pathogenesis during hepatitis B virus (HBV) infection. Our previous study found that CD205⁺ macrophages in the liver of hepatitis B surface antigen transgenic (HBs-Tg) mice increased significantly compared with those in wild-type mice, and these increased CD205⁺ macrophages were involved in CpG-oligodeoxynucleotide-induced liver injury in HBs-Tg mice. Here, we analysed the phenotype and function of CD205⁺ macrophages derived from the liver of HBs-Tg mice and patients with chronic hepatitis B (CHB). We found that HBs-Tg mice-derived hepatic macrophages produced larger amounts of pro-inflammatory cytokines, including IL-6, IL-12, TNF-α, and of the anti-inflammatory cytokine IL-10 after stimulation with CpG-oligodeoxynucleotides or commensal bacteria DNA than B6 mice-derived hepatic macrophages. Furthermore, hepatic CD205⁺ macrophages from HBs-Tg mice showed an activated phenotype and expressed higher levels of inflammatory cytokine genes, chemokine genes, and phagocytosis-related genes than hepatic CD205^- macrophages. In addition, CD205⁺ macrophages displayed an inflammatory phenotype and were increased in the liver of patients with CHB compared with those in healthy controls. Our data suggest that hepatic CD205⁺ macrophages are a unique pro-inflammatory subset observed during HBV infection. Thus, development of intervention targeting these cells is warranted for immunotherapy of HBV-induced liver diseases.

Figure 1. Inflammatory cytokines produced by hepatic macrophages in HBs-Tg and B6 mice.

Hepatic macrophages were isolated from HBs-Tg (n = 5) and B6 (n = 5) mice and enriched using EasySep^TM Mouse Monocyte Enrichment Kit. Cells were stimulated with CpG (1 μM) or E. coli DNA (50 μg/mL) for 24 hours. Cytokines were measured by ELISA. Data represent mean ± SD from two independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed unpaired Student’s t-test.

Figure 2. Expression of activation makers on hepatic macrophages in HBs-Tg mice.

(a) Expression of CD11b, Ly6C, CD14, CD40, CD80, and CD86 on hepatic CD205^+/− macrophages in HBs-Tg mice (n = 4). (b) Statistical analysis of mean fluorescence intensity (MFI) of the markers shown in (a). Data are expressed as mean ± SD, and p values were calculated using two-tailed unpaired Student’s t-test. *p < 0.05, **p < 0.01 for the indicated comparisons.

Figure 3. Cytokine gene expression profiles of hepatic CD205⁺ and CD205⁻ macrophages.

Hepatic macrophage subsets were isolated from HBs-Tg mice by FACS sorting. Quantitative PCR analysis compared mRNA expression levels of cytokine genes between hepatic CD205⁺ and CD205⁻ macrophages. Data are normalised to Actb in the same sample and expressed as fold change in comparison with CD205⁻ macrophages. Data represent mean ± SD from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed unpaired Student’s t-test.

Figure 4. Expression of chemokine and chemokine receptor genes in hepatic CD205⁺ and CD205⁻ macrophages.

Quantitative PCR analysis of relative mRNA levels of chemokine genes (a) and chemokine receptor genes (b) in hepatic CD205⁺ and CD205⁻ macrophages derived from HBs-Tg mice. Values are expressed as fold amplification over CD205⁻ macrophages following normalization with Actb. Data represent mean ± SD from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed unpaired Student’s t-test.

Figure 5. Hepatic CD205⁺ macrophages in HBs-Tg mice overexpress phagocytosis-related genes.

Quantitative PCR analysis comparing mRNA levels of phagocytosis-related genes between hepatic CD205⁺ and CD205⁻ macrophages in HBs-Tg mice. Values are expressed as fold amplification over CD205⁻ macrophages following normalization with Actb. Data represent mean ± SD from three independent experiments. *p < 0.05, **p < 0.01, two-tailed unpaired Student’s t-test.

Figure 6. Percentage of peripheral CD205⁺ monocytes is markedly increased in HBsAg-positive donors.

(a) Monocyte subsets in peripheral blood were gated based on forward and side scatter (left panel), and CD205 expression on peripheral monocytes of HBsAg-positive donors (HPD) and healthy controls (HC) (right panel). (b) Pooled data show the percentages of CD205⁺ monocyte subset in HPD (n = 36) and HC (n = 36). Data are expressed as mean ± SD. **p < 0.01, two-tailed unpaired Student’s t-test.

Figure 7. CD205⁺ macrophages are increased in the liver of patients with CHB.

(a) A representative graph of hepatic CD68⁺CD205^+/− macrophages in patients with CHB and healthy controls (HC). (b) Individual values of proportions of hepatic CD68⁺CD205^+/− macrophages in patients with CHB (n = 8) and HC (n = 5). Data are expressed as mean ± SD, and p values were calculated using two-tailed unpaired Student’s t-tests. **p < 0.01. (c) Immunofluorescence microscopy images of human livers from two patients with CHB and two HC stained with anti-CD205 (green), anti-CD68 (red), and DAPI (blue). The solid arrows show CD68⁺CD205⁺ macrophages, and the dashed arrows show CD68⁺CD205⁻ macrophages.

Figure 8. Identification of CD205⁺ macrophages in the liver of patients with CHB.

(a,b) Expression of CD14, CD16, and HLA-DR on hepatic CD205^+/− macrophages derived from patients with CHB (n = 4). (c,d) Hepatic leukocytes were stimulated with PMA (30 ng/mL) and ionomycin (1 μg/mL) for 4 hours in the presence of monensin. Intracellular expression levels of TNF-α and IL-12/23p40 in hepatic CD205⁺ and CD205⁻ macrophages derived from patients with CHB (n = 5) were detected by flow cytometry. Data are expressed as mean ± SD. *p < 0.05, **p < 0.01, two-tailed unpaired Student’s t-test.