Characterization of Liver Monocytic Myeloid-Derived Suppressor Cells and Their Role in a Murine Model of Non-Alcoholic Fatty Liver Disease

doi:10.1371/journal.pone.0149948

Fig 1.

CD11b⁺Gr1⁺ cells in the livers of normal mice and high-fat-diet mice were comprised of 3 subsets.

(A) Frequency of CD11b⁺Gr1⁺ cells in mouse liver non-parenchymal cells (NPCs) at 3 months (3 m) or 12 months (12 m) of age (n = 5). *P < 0.05 (B) Analysis of CD11b⁺Gr1⁺ cells by the side-scatter (SSC) of light during flow cytometry. (C) Frequency of CD11b⁺Gr1^high, SSC^lowCD11b⁺Gr1^dim, and SSC^high CD11b⁺Gr1^dim cells in mouse liver NPCs at 3 m or 12 m of age (n = 5). *P < 0.05 compared to frequency of each cells at 3 months. ND, normal diet; HFD, high fat diet.

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Fig 2.

Characterization of liver CD11b⁺Gr1⁺ cell subsets.

(A) Representative histograms of the phenotypic profiles of liver CD11b⁺Gr1^high, SSC^highCD11b⁺Gr1^dim, and SSC^lowCD11b⁺Gr1^dim cells. Closed histograms represent background control staining; open histograms represent staining by indicated monoclonal antibody (mAb). The percentage of positive cells is indicated. (B) Morphology of purified liver CD11b⁺Gr1^high, SSC^highCD11b⁺Gr1^dim, and SSC^lowCD11b⁺Gr1^dim cells by Wright-Giemsa staining (100× magnification). Scale bars, 20 μm. SSC, light side scatter. The sizes of SSC^highCD11b⁺Gr1^dim and SSC^lowCD11b⁺Gr1^dim cells are shown in the right panel. *P < 0.05

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Fig 3.

SSC^lowCD11b⁺Gr1^dim cells in the livers suppress T cell responses.

(A) Proliferation of CFSE-labeled T cells cultured in the presence of Dynabeads mouse T-Activator CD3/CD28 with or without liver SSC^high or SSC^low CD11b⁺Gr1^dim cells. (B) Representative image of intracellular interferon-γ staining for T cells cultured with or without liver SSC^high or SSC^low CD11b⁺Gr1^dim cells. (C) T cells and allogenic dendritic cells were co-cultured. Liver SSC^high and SSC^lowCD11b⁺Gr1^dim cells were added to the cultures. The data obtained from 3 separate experiments are shown. *P < 0.05 compared to the levels of T cell proliferation in allogenic mixed lymphocyte reactions (aMLRs) without CD11b⁺Gr1^dim cells.

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Fig 4.

The suppressive function of liver SSC^lowCD11b⁺Gr1^dim cells is dependent on a NO mechanism.

(A) Proliferation of carboxy-fluorescein diacetate, succinimidyl ester (CFSE)-labeled T cells cultured in the presence of Dynabeads mouse T-Activator CD3/CD28 along with purified liver SSC^lowCD11b⁺Gr1^dim cells. Different enzyme inhibitors (L-NIL, nor-NOHA, or catalase) were added at the start of each respective culture. (B) Nitrite levels were investigated in the culture supernatants after 60 h of co-culture (n = 3). (C) The intracellular iNOS expression was determined by flow cytometry. The percentage of positive cells is indicated. The data obtained from 3 separate experiments are shown. *P < 0.05 compared to T cells alone.

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Fig 5.

The CCL2/CCR2 pathway mediates the migration of SSC^lowCD11b⁺Gr1^dim cells to the NAFLD liver.

(A) CCL2 expression in the livers of ND and HFD-fed mice was investigated by real-time RT-PCR (n = 5). (B, C) Protein expression of CCL2 in the livers was investigated by ELISA (B) (n = 5) and immunohistochemistry (C). Scale bars, 100 μm. (D) CCL2 gene expression in Hepa1-6 cells treated with oleic acid or palmitic acid analyzed by real-time RT-PCR (n = 5). (E) Migration assays revealed that SSC^lowCD11b⁺Gr1^dim cells migrated in response to CCL2 in vitro (n = 5). *P < 0.05

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Fig 6.

M-CSF is associated with SSC^lowCD11b⁺Gr1^dim cell expansion in the liver.

(A) Liver M-CSF mRNA was investigated by real-time RT-PCR (n = 5). (B) Liver M-CSF protein was investigated by immunohistochemistry (n = 5). Scale bars, 100 μm. (C) M-CSF mRNA in Hepa1-6 cells treated with fatty acid was analyzed by real-time RT-PCR (n = 5). (D) Bone marrow cells were cultured with M-CSF. The frequency of CD11b⁺Gr1^dimLy6C^high cells among the CD11b⁺Gr1^dim cells is shown. (E) The Ly6C^high and Ly6C^low populations (S1 Fig) were sorted and added to allogenic MLRs. *P < 0.05

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