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, 69 (3), 676-686

Macrophages Contribute to the Pathogenesis of Sclerosing Cholangitis in Mice

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Macrophages Contribute to the Pathogenesis of Sclerosing Cholangitis in Mice

Maria Eugenia Guicciardi et al. J Hepatol.

Abstract

Background & aims: Macrophages contribute to liver disease, but their role in cholestatic liver injury, including primary sclerosing cholangitis (PSC), is unclear. We tested the hypothesis that macrophages contribute to the pathogenesis of, and are therapeutic targets for, PSC.

Methods: Immune cell profile, hepatic macrophage number, localization and polarization, fibrosis, and serum markers of liver injury and cholestasis were measured in an acute (intrabiliary injection of the inhibitor of apoptosis antagonist BV6) and chronic (Mdr2-/- mice) mouse model of sclerosing cholangitis (SC). Selected observations were confirmed in liver specimens from patients with PSC. Because of the known role of the CCR2/CCL2 axis in monocyte/macrophage chemotaxis, therapeutic effects of the CCR2/5 antagonist cenicriviroc (CVC), or genetic deletion of CCR2 (Ccr2-/- mice) were determined in BV6-injected mice.

Results: We found increased peribiliary pro-inflammatory (M1-like) and alternatively-activated (M2-like) monocyte-derived macrophages in PSC compared to normal livers. In both SC models, genetic profiling of liver immune cells identified a predominance of monocytes/macrophages; immunohistochemistry confirmed peribiliary monocyte-derived macrophage recruitment (M1>M2-polarized), which paralleled injury onset and was reversed upon resolution in acute SC mice. PSC, senescent and BV6-treated human cholangiocytes released monocyte chemoattractants (CCL2, IL-8) and macrophage-activating factors in vitro. Pharmacological inhibition of monocyte recruitment by CVC treatment or CCR2 genetic deletion attenuated macrophage accumulation, liver injury and fibrosis in acute SC.

Conclusions: Peribiliary recruited macrophages are a feature of both PSC and acute and chronic murine SC models. Pharmacologic and genetic inhibition of peribiliary macrophage recruitment decreases liver injury and fibrosis in mouse SC. These observations suggest monocyte-derived macrophages contribute to the development of SC in mice and in PSC pathogenesis, and support their potential as a therapeutic target.

Lay summary: Primary sclerosing cholangitis (PSC) is an inflammatory liver disease which often progresses to liver failure. The cause of the disease is unclear and therapeutic options are limited. Therefore, we explored the role of white blood cells termed macrophages in PSC given their frequent contribution to other human inflammatory diseases. Our results implicate macrophages in PSC and PSC-like diseases in mice. More importantly, we found that pharmacologic inhibition of macrophage recruitment to the liver reduces PSC-like liver injury in the mouse. These exciting observations highlight potential new strategies to treat PSC.

Keywords: C-C chemokine ligand 2 (CCL2); C-C chemokine receptor 2 (CCR2); Cenicriviroc (CVC); Cholestatic liver injury; Liver fibrosis; Macrophages; Sclerosing cholangitis.

Conflict of interest statement

Conflict of interest: At the time of the study, P.V. was an employee of Tobira (subsequently acquired by Allergan), which provided the CCR2/CCR5 antagonist cenicriviroc. All other authors have declared no conflict of interest related to this study.

Figures

Fig. 1
Fig. 1. Macrophages accumulate in the peribiliary areas of the livers of PSC patients
(A) CD68 immunohistochemistry on healthy and cirrhotic-stage PSC livers (20×); quantification of CD68+ area. Arrows: bile ducts. (B) CD68 (total macrophages), CK19 (cholangiocytes) co-immunofluorescence (40×); quantification of CD68+ cells (n=4 normal, n=3 PSC). (C) CD68, CK19 and CCR2 (recruited macrophages) co-immunofluorescence (40×); quantification of CCR2+ cells (n=3/group) (D) CK19, iNOS (pro-inflammatory macrophages) and CD206 (anti-inflammatory macrophages) co-immunofluorescence (63×); quantification of iNOS+ and CD206+ cells (4 normal; 3 PSC). Higher magnifications of the dotted areas are shown in the lower row. *p<0.05; **p<0.005 (Mann-Whitney).
Fig. 2
Fig. 2. Expression of monocyte/macrophage-associated genes is enhanced in the liver of two mouse models of sclerosing cholangitis
Prevalence of immune cells in the liver of (A) BV6-treated vs. saline-injected C57BL/6 and (B) C57BL/6.Mdr2−/− vs. wild-type mice evaluated by gene expression using NanoString technology. The graphs depict genes significantly over-expressed (p<0.05) in BV6-treated wild-type mice or Mdr2−/− mice by a minimum of two-fold above that of saline-injected wild-type or wild-type mice, respectively. The genes were attributed to immune cell types and hierarchically ranked by number of over-expressed genes.
Fig. 3
Fig. 3. Macrophages accumulate in the peribiliary areas of BV6-injected mouse livers
(A) CD68 liver immunohistochemistry on BV6- or saline-injected mice at day 5 (20×); quantification of CD68+ (saline n=5; BV6 n=4; 3–6 areas/sample). (B) CD68 liver immunohistochemistry on untreated mice (normal) or BV6-injected mice at day 3, 5, 7 or 21 (20×); quantification of CD68+ area (normal, day 7: n=3; day 3, day 5: n=4; day 21: n=5; 3–6 areas/sample). (C) CD68 liver immunohistochemistry on sixty-day old wild-type and C57BL/6.Mdr2−/− female mice (10×). (D)(E) iNOS, CD206 and EpCAM or CK19 (cholangiocytes) liver co-immunofluorescence of (D) saline- or BV6-injected mice (day 5) and (E) sixty-day old wild-type and C57BL/6.Mdr2−/− female mice; quantification of iNOS+ and CD206+ cells (BV6 n=3/group; wild type n=4 females; Mdr2−/− n=3 females). (F) Macrophage and cholangiocyte co-immunofluorescence of BV6-treated (left), Mdr2−/− (center) and PSC livers (right). Arrows: macrophages. ***p<0.001; ****p<0.0001 (Mann-Whitney).
Fig. 4
Fig. 4. Macrophages accumulate in the peribiliary areas of Mdr2−/− mouse livers
(A) Clec4F (KC), CCR2 and CK19 liver co-immunofluorescence on normal and BV6-injected mice (day 3–21); quantification of Clec4F+ and CCR2+ cells (n=3/group). (B) Representative flow cytometric plots showing CD45+F4/80+ liver macrophage population, CD45+F4/80+CD11bhi monocyte-derived macrophages (MoMF), CD45+F4/80+CD11bint Kupffer cells (KC) and CD45+F4/80+Ly6Chi inflammatory macrophages in the liver of saline- or BV6-injected mice (day 5), and sixty-day old C57BL/6.Mdr2−/− mice. Quantification shown on the right (3 saline, 5 BV6, 4 C57BL/6, 4 Mdr2−/−). *p<0.05 (Mann-Whitney).
Fig. 5
Fig. 5. Active and senescent cholangiocytes release monocyte chemoattractants and macrophage-activating factors
(A) ELISA on supernatants of H69 cells treated with DMEM + BV6 (n=3 in quintuplicate). (B) Transcriptomic profiles of H69, NHC and isolated PSC cholangiocytes by high-throughput NGS. (C) ELISA on supernatants from isolated PSC cholangiocytes (n=3) or NHC + LPS (n=3). RPKM: reads/kilobase of transcript/million mapped reads. (D) THP-1 cell transmembrane migration following exposure for 6 hours to conditioned media as in (A), or (C), ± neutralizing IL-8 or CCL2 antibodies. THP-1 exposed to DMEM used as controls (DMEM)(BV6 n=3; NHC+LPS n=3; PSC n=4 at different times from PSC-1). RFU: relative fluorescent units. (E) THP-1 cell migration in a dual-chamber microfluidic device after 24 hr. co-culture (PSC1, black symbols, PSC2, grey symbols, n=3 each). (F) Gene expression in THP-1 cells incubated with conditioned media as in (A) or (C) (PSC1, black symbols, n=6; PSC2, grey symbols, n=3). *p<0.05; **p<0.005; ***p<0.0005; ****p<0.0001 (Mann-Whitney).
Fig. 6
Fig. 6. Pharmacologic inhibition of CCR2-dependent monocyte recruitment attenuates biliary injury
(A) Liver sections of BV6-injected mice (5 days) ± CVC or vehicle (20×). Arrows: bile ducts. pv: portal vein. Quantification of CD68+ cells on the right. (B) Liver hydroxyproline content. (C) Serum ALT, total BA and total bilirubin. (D) Macrophage infiltration and hepatic fibrosis markers by gene expression in whole livers. BV6 (n=8), BV6+vehicle (n=11), BV6+CVC (n=9). (E) Representative flow cytometric plots and quantification of CD45+CCR2+, CD45+CCR2+F4/80+ (macrophages) and CD45+CCR2+CD3+ (lymphocytes) liver cells. Saline (n=3); BV6 (n=5). (F) Ccr5 and Ccl5/Rantes gene expression. *p<0.05; **p<0.005 (Mann-Whitney).
Fig. 7
Fig. 7. Genetic disruption of the CCR2/CCL2 axis attenuates biliary injury
(A) Liver sections of saline- or BV6-injected wild-type and Ccr2−/− mice at day 5 (20×). Arrows: bile ducts. pv: portal vein. (B) Liver hydroxyproline content. (C) ALT, total BA and total bilirubin. (D) Macrophage infiltration markers and fibrosis markers by gene expression in whole livers. Wild-type: saline (n=8), BV6 (n=8); Ccr2−/−: saline (n=4), BV6 (n=5). *p<0.05, **p<0.005 (Mann-Whitney).

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