Bone marrow-derived macrophages distinct from tissue-resident macrophages play a pivotal role in Concanavalin A-induced murine liver injury via CCR9 axis

The fundamental mechanism how heterogeneous hepatic macrophage (Mφ) subsets fulfill diverse functions in health and disease has not been elucidated. We recently reported that CCR9+ inflammatory Mφs play a critical role in the course of acute liver injury. To clarify the origin and differentiation of CCR9+Mφs, we used a unique partial bone marrow (BM) chimera model with liver shielding for maintaining hepatic resident Mφs. First, irradiated mice developed less liver injury with less Mφs accumulation by Concanavalin A (Con A) regardless of liver shielding. In mice receiving further BM transplantation, CD11blowF4/80high hepatic-resident Mφs were not replaced by transplanted donors under steady state, while under inflammatory state by Con A, CCR9+Mφs were firmly replaced by donors, indicating that CCR9+Mφs originate from BM, but not from hepatic-resident cells. Regarding the mechanism of differentiation and proliferation, EdU+CCR9+Mφs with a proliferative potential were detected specifically in the inflamed liver, and in vitro study revealed that BM-derived CD11b+ cells co-cultured with hepatic stellate cells (HSCs) or stimulated with retinoic acids could acquire CCR9 with antigen-presenting ability. Collectively, our study demonstrates that inflammatory Mφs originate from BM and became locally differentiated and proliferated by interaction with HSCs via CCR9 axis during acute liver injury.


Isolation of tissue immune cells
Intestinal lamina propria mononuclear cells were separated as described previously 1 .
Mesenteric lymph nodes and Peyer's patch were minced and passed through 100-μm nylon mesh. BM and peripheral blood (PB) leukocytes were hemolyzed and passed through 40-μm nylon mesh. The epididymal adipose tissue stromal vascular fraction (SVF) was prepared by digestion with 2 mg/mL collagenase type II (Sigma-Aldrich) digestion for 20 minutes. After centrifugation, the cell pellet was harvested as the SVF including adipose tissue leukocytes.
Liver and spleen mononuclear cells were isolated as described previously 2 . Briefly, livers were perfused through the portal vein and then minced and passed through 100-μm nylon mesh. The filtrate was centrifuged at 100 × g for 1 minute to eliminate hepatocytes with debris, and the supernatant was washed once. The cells were suspended in Hanks' balanced salt solution and overlaid on Histopaque solution (Sigma-Aldrich). After specific gravity centrifugation at 780 × g for 20 minutes, the cells were collected from the upper face of the Histopaque solution and subjected to flow cytometry. To determine the origin of CCR9 + Mφs, liver-resident and recruited Mφs were separated as described previously 3 with slight modifications. Briefly, livers were minced and filtered without any enzymatic digestion. The resulting cells were resuspended in 25% Percoll solution (GE Healthcare UK, Buckinghamshire, England) and gradient-centrifuged with 50% Percoll solution.

Flow cytometry analysis
After blocking with an anti-FcR antibody (CD16/32; BD Pharmingen, San Diego, CA) for 20 minutes at 4°C, cells were incubated with specific fluorescence-labeled monoclonal antibodies at 4°C for 30 minutes. The following monoclonal antibodies

Splenectomy
The spleen was removed surgically under deep anesthesia. Briefly, after skin sterilization, a left-flank incision of about 5 mm was made to expose the spleen and the whole spleen was gently removed after astriction. The peritoneum and skin were separately closed with sutures. In control mice, a sham operation was performed as well as a splenectomy procedure without removing the spleen. The mice were housed for 2 S4 weeks after surgery to allow healing and then subjected to experiments.

Hepatic resident macrophages depletion with Clodronate liposomes
In order to deplete hepatic resident macrophage, mice were injected with 200μL of Clodronate liposomes or control liposomes (FormuMax Scientific Inc., Sunnyvale, CA) 24 hours prior to Con A administration. Liver mononuclear cells extracted at 12 hours following Con A administration were analyzed by flow cytometry.

In vivo EdU uptake study
EdU labeling was performed using a Click-iT® Plus EdU Cytometry Assay Kit (Molecular Probes). Briefly, 1 mg/head EdU was intraperitoneally injected into WT mice at 10 hours after administration of Con A or PBS and allowed to incorporate into newly synthesized DNA for fluorescent labeling for 2 hours in vivo. At 2 hours after EdU injection, the liver and PB were collected and the cells were analyzed by flow cytometry.

Preparation of tissue extracts
The collected livers and spleens were minced, suspended in RPMI 1640 medium, and passed through 100-μm nylon mesh. The cell suspensions were sufficiently sonicated while cooling and debris was removed by centrifugation, followed by 0.22-μm polyvinylidene difluoride membrane filtration. The filtrates were collected and the total protein concentrations were measured using a BCA Protein Assay Kit (Pierce, Rockford, IL). The extracts were subdivided and stored at −80°C until use in experiments.

Isolation of HSCs and liver sinusoidal endothelial cells (LSECs)
HSCs and LSECs were separated as described previously 4 . Briefly, cells were isolated by collagenase and protease perfusion into the portal vein, followed by 8.2% and 17% Nycodenz (Accurate Chemical and Scientific Corporation, Westbury, NY) three-layer discontinuous density-gradient centrifugation. HSCs were collected from the upper face of the 8.2% Nycodenz and LSECs were harvested from the upper face of the 17% Nycodenz. The cell extracts were prepared as described above for the tissue extracts.

In vitro differentiation assay
Freshly harvested total BM cells were seeded at 5.0 × 10 5 cells in 96-well plates and cultured with cell extracts (7 mg/mL each) for 6 hours. The cells were then harvested, washed, and analyzed by flow cytometry.

In vitro co-culture study
After isolation of naïve BM cells (CD45.1) and HSCs from PBS or Con A treated mice liver (CD45.2), 1.7 × 10 6 cells of HSCs and 5 × 10 5 cells of total BM cells were co-cultured for 4 days in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and 1% penicillin/streptomycin. The cells were then harvested, washed, and CD45.1 positive cells were analyzed by flow cytometry.

Fluorescence immunohistochemistry
Livers were perfused and isolated from Con A-treated mice. Part of each liver was excised, embedded in OCT Compound (Sakura Finetek, Torrance, CA) after excluding moisture, and sliced onto slide glasses (Matsunami, Osaka, Japan). The sliced sections After washout of excess antibodies, the sections were subjected to mounting and nuclear staining using VECTASHIELD with DAPI (Vector Laboratories, Burlingame, CA).
Analysis was performed using a TCS SP5 confocal microscope (Leica, Jena, Germany).