B Cell-Mediated Maintenance of Cluster of Differentiation 169-Positive Cells Is Critical for Liver Regeneration

Abstract

The liver has an extraordinary capacity to regenerate through activation of key molecular pathways. However, central regulators controlling liver regeneration remain insufficiently studied. Here, we show that B cell-deficient animals failed to induce sufficient liver regeneration after partial hepatectomy (PHx). Consistently, adoptive transfer of B cells could rescue defective liver regeneration. B cell-mediated lymphotoxin beta production promoted recovery from PHx. Absence of B cells coincided with loss of splenic cluster of differentiation 169-positive (CD169⁺ ) macrophages. Moreover, depletion of CD169⁺ cells resulted in defective liver regeneration and decreased survival, which was associated with reduced hepatocyte proliferation. Mechanistically, CD169⁺ cells contributed to liver regeneration by inducing hepatic interleukin-6 (IL-6) production and signal transducer and activator of transcription 3 activation. Accordingly, treatment of CD169⁺ cell-depleted animals with IL-6/IL-6 receptor rescued liver regeneration and severe pathology following PHx. Conclusion: We identified CD169⁺ cells to be a central trigger for liver regeneration, by inducing key signaling pathways important for liver regeneration.

Figure 1

Decreased liver regeneration in splenectomized and B cell–deficient mice following PHx. (A) Survival of splenectomized, 70% PHx, and splenectomized mice followed by PHx (PHx+S) was monitored (n = 14‐19). (B) The liver weight/body weight ratio was determined at the indicated time points in WT sham‐operated mice and splenectomized mice (left panel) and in PHx WT mice and splenectomized mice (PHx+S) (right panel) (n = 3‐5). (C,D) Sections of snap‐frozen liver tissue from 70% PHx and splenectomized mice followed by PHx (PHx+S) at the indicated time points were stained with (C) anti‐phospho‐H3 and (D) anti‐Ki‐67 antibodies. Representative sections for each time point are shown (n = 4; scale bar, 100 μm). Right panels indicate quantification. (E) B‐cell numbers were determined by flow cytometry in the newly regenerated (“New,” n = 7‐8) and remaining (“Old,” n = 3‐4) liver lobes and spleen tissue (n = 7‐8) at indicated time points after 70% PHx. Results were calculated according to the liver (grams) and spleen (milligrams) weights. (F) Survival of Jh^–/– mice (n = 9) after 70% PHx compared to sham‐operated Jh^–/– mice (n = 3) and WT mice (n = 6). Error bars in all experiments represent SEM; *P < 0.05,  **P < 0.01, ***P < 0.001. Abbreviation: S, splenectomy.

Figure 2

B cells play a crucial role in liver regeneration after PHx. (A) The activity of AST and ALT was measured in serum of WT and Jh^–/– mice following PHx at the indicated time points (n = 4‐5). (B) Sections of snap‐frozen liver tissue from WT and Jh^–/– mice following PHx were stained with H&E. One representative set of n = 3 is shown (scale bar, 200 μm). (C) Survival of Baffr^–/– mice (n = 18) after 70% PHx compared to sham‐operated Baffr^–/– mice (n = 4) and WT mice after 70% PHx (n = 7) was monitored. (D) The activity of AST and ALT was measured in serum of WT and Baffr^–/– mice following PHx at the indicated time points (n = 4‐5). (E,F) Sections of snap‐frozen liver tissue from WT and Jh^–/– mice following PHx were stained with (E) anti‐phospho‐H3 and (F) anti‐Ki‐67 antibodies. Representative sections for each time point are shown (n = 3; scale bar, 100 μm). Lower panels indicate quantification. Error bars in all experiments represent SEM; *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

B cell–derived Ltβ contributes to liver regeneration after PHx. (A) RNA expression of Ltβ was measured in spleen and liver tissue from WT and Jh^–/– mice at the indicated time points post–70% PHx (n = 3‐4). (B) Protein level of Ltβ was measured in liver tissue from naive WT and Jh^–/– and from WT and Jh^–/– mice at the indicated time points post–70% PHx. (C‐E) Purified B cells (2 × 10⁶) from WT mice were intravenously injected into Jh^–/– mice. After 48 hours, (C) B‐cell numbers were determined in the spleen by flow cytometry (n = 4‐6). (D) RNA levels of Ltβ were measured in liver tissue (n = 3‐5). (E) The protein level of Ltβ was measured in liver tissue. (F) Survival of Jh^–/– mice without (n = 6) or after (n = 7) B‐cell transfer was determined following 70% PHx. (G) Survival of Baffr^–/– mice without (n = 7) or after (n = 7) B‐cell transfer was determined following 70% PHx. (H) Survival of untreated (n = 13) and agonist LtβR antibody–treated (n = 13) Jh^–/– mice was monitored after 70% PHx. Error bars in all experiments represent SEM; *P < 0.05, **P < 0.01.

Figure 4

CD169⁺ cells contribute to liver regeneration. (A) Sections of snap‐frozen spleen tissue from WT and Jh^–/– mice were stained with anti‐CD169, anti‐B220, and anti‐CD90.2 antibodies. Representative sections are shown (n = 4‐5; scale bar, 100 μm). Right panel indicates average and SEM of mean fluorescence intensities of CD169 staining. (B) Sections from snap‐frozen spleen tissue from WT and Baffr^–/– mice were stained with anti‐CD169 anti‐B220 and anti‐CD90.2 antibodies. Representative sections are shown (n = 4‐5; scale bar, 100 μm). Right panel indicates average and SEM of mean fluorescence intensities of CD169 staining. (C) Purified B cells (2 × 10⁶) from WT mice were adoptively transferred into Jh^–/– mice. After 48 hours, CD169‐cell numbers were determined in the spleen by flow cytometry (n = 4‐6). (D,E) CD169⁺ cells were measured by flow cytometry in the newly regenerated (n = 7‐8) and remaining (“Old”) (n = 3‐4) liver lobes (D) and spleen tissue (n = 7‐8) (E) at the indicated time points after 70% PHx. Results were calculated according to liver (grams) and spleen (milligrams) weights. (F) The activity of AST and ALT was measured in serum of WT and DT‐treated CD169‐DTR mice following PHx at the indicated time points (n = 3). (G) Survival of DT‐treated CD169‐DTR mice after PHx (n = 12) compared to WT mice (n = 8) after PHx, CD169‐DTR mice (n = 5) after PHx, and sham‐operated, DT‐treated CD169‐DTR mice (n = 4) was monitored. Error bars in all experiments represent SEM; *P < 0.05, **P < 0.01, ****P < 0.0001. Abbreviations: DAPI, 4′,6‐diamidino‐2‐phenylindole; MFI, mean fluorescence intensity.

Figure 5

CD169⁺ cells promote the presence of hepatic proliferation markers after PHx. (A) The liver weight/body weight ratio was determined after 70% PHx in DT‐treated WT mice and DT‐treated CD169‐DTR mice followed by PHx (n = 3). (B,C) Sections of snap‐frozen liver tissue from CD169‐DTR and DT‐treated CD169‐DTR mice at the indicated time points after 70% PHx were stained with anti‐phospho‐H3 (B) and anti‐Ki‐67 (C) antibodies. Representative sections for each time point are shown (n = 3; scale bar, 100 μm). Right panels indicate quantification. (D) Protein level of PCNA was measured at the indicated time points after 70% PHx in DT‐treated CD169‐DTR and CD169‐DTR mice (n = 4). Lower panel indicates quantification. (E) Liver weight (left panel) and liver morphology (right panel) were determined at day 13 after 70% PHx of DT‐treated CD169‐DTR and CD169‐DTR mice (n = 3‐5). Error bars in all experiments represent SEM; *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6

Defective activation of IL‐6 signaling in the absence of CD169⁺ cells. (A) RNA expression level of cytokines important for liver regeneration was determined in the liver tissue from WT and Jh ^–/– mice at the indicated time points post–70% PHx (n = 3). (B) Protein expression of IL‐6 was determined in liver tissue from WT and Jh ^–/– mice at the indicated time points after 70% PHx (n = 3). Lower panel indicates quantification. (C) RNA expression levels of IL‐6 was measured in the liver tissue of WT and Jh ^–/– mice without or with purified B cells (n = 3‐5). (D,E) RNA expression levels of Il‐6 were determined in liver tissue from DT‐treated WT, CD169‐DTR, and DT‐treated CD169‐DTR mice as labeled at the indicated time points after 70% PHx (n = 3‐5). (F) Protein expression of IL‐6 was determined in liver tissue from CD169‐DTR and DT‐treated CD169‐DTR mice at the indicated time points after 70% PHx (n = 3). Lower panel indicates quantification. (G) Protein lysates of liver tissue from CD169‐DTR mice and DT‐treated CD169‐DTR mice at the indicated time points after PHx were blotted and stained with anti‐phospho‐Erk, anti‐Erk, anti‐phospho‐STAT3, anti‐STAT3, anti‐IκBα, and anti‐β‐actin antibodies (one representative of n = 3 blots is shown). Error bars in all experiments represent SEM; *P < 0.05, **P < 0.01, ***P < 0.001. Abbreviations: Amphr, amphiregulin; Hbegf, heparin‐binding EGF‐like growth factor; Hgf, hepatocyte growth factor; Fgf, fibroblast growth factor; Pdgf, platelet‐derived growth factor; Vegf, vascular endothelial growth factor.

Figure 7

IL‐6/IL‐6R treatment can rescue liver regeneration in the absence of CD169⁺ cells. (A) Protein lysates of liver tissue from CD169‐DTR mice or DT‐treated CD169‐DTR mice following IL‐6/IL‐6R treatment were blotted and stained with anti‐phospho‐Erk, anti‐Erk, anti‐phospho‐STAT3, anti‐STAT3, anti‐IκBα, and anti‐β‐actin antibodies. One representative of n = 6 blots is shown. Right panels indicate quantification. (B) The liver weight/body weight ratio was determined at 48 hours after 70% PHx in CD169‐DTR mice and DT‐treated CD169‐DTR mice and following IL‐6/IL‐6R treatment (n = 3‐5). (C,D) Sections of snap‐frozen liver tissue from DT‐treated CD169‐DTR mice without or after IL‐6/IL‐6R treatment at 48 hours after 70% PHx were stained with (C) anti‐phospho‐H3 and (D) anti‐Ki‐67 antibodies. Representative sections for each time point are shown (n = 3‐5; scale bars, [C] 50 μm, [D], 100 μm). Right panels indicate quantification. (E) Protein level of PCNA was measured in DT‐treated CD169‐DTR mice in the absence and presence of IL‐6/IL‐6R treatment at 48 hours after 70% PHx (n = 5). Lower panel indicates quantification. (F) Survival of DT‐treated CD169‐DTR mice without or after IL‐6/IL‐6R treatment was monitored (n = 8‐9). Error bars in all experiments represent SEM; *P < 0.05, **P < 0.01, ***P < 0.001.