Fig 1.
Distinct liver myeloid cell subsets are sequentially mobilized in T. congolense-infected mice.
Ly6C+ monocytes (Ly6C+ Mo), Ly6C- monocytes (Ly6C- Mo) and macrophages (MF) were gated based on Ly6C and CX3CR1/GFP expression as illustrated in S1A Fig in S1 Text in non-infected (n.i) and infected CX3CR1-GFP+/- mice at day 7, 14 and 21 pi. Numbers of (A) the 3 myeloid cell subsets and (B) of the MHC-II- to lo and MHC-IIint to hi fraction of Ly6C+ monocytes, Ly6C- monocytes and macrophages within liver non-parenchymal cell population were determined. Data are shown as mean + SD of 3 individual mice from one representative out of four independent experiments. * p< 0.05 compared to non-infected mice; § p<0.05 comparing populations linked by horizontal bar. (C) F4/80 or CD115 expression on Ly6C+ monocytes, Ly6C- monocytes and macrophages from the blood or the liver at day 7 and 14 pi determined as mean fluorescence intensity difference (dMFI) between anti-F/80 or anti-CD115 and isotype control antibodies.
Fig 2.
Ly6C+ monocytes differentiate into macrophages in T. congolense-infected mice.
(A) Liver GFP+ Ly6C+ monocytes (Ly6C+ Mo) purified from CD45.2 LysM-GFP mice at day 7 pi were transferred in CD45.1 WT mice at d8 pi. After 15, 36 and 72 h, liver CD45.2+ GFP+ cells from recipient mice were analysed for CD11b and Ly6C expression. MHC-II, F4/80, Mertk, CD64, CD11c and Mar1 expression was then investigated in Ly6C- CD11b+ cells. FACS profiles are representative of 1 out of 9 mice tested in three independent experiments. Percentages of cells in indicated gates are as shown as mean ± SD of 3 individual mice of one representative out of three independent experiments. (B) ALT levels in mice treated with Ly6C+ monocytes or with HBSS as control. Data are shown as mean + SD of 3 individual mice of one representative out of three independent experiments. * p<0.05 compared to control mice.
Fig 3.
Ly6C+ monocytes are the main producers of TNF while Ly6C- monocytes and macrophages are the major source of IL-10 in T. congolense-infected mice.
(A) Intracellular TNF+ cells were gated in liver non-parenchymal cells from CX3CR1-GFP+/- mice at day 7 and 21 pi. The percentage of Ly6C+ monocytes (Ly6C+ Mo), Ly6C- monocytes (Ly6C- Mo), macrophages (MF) gated as in Fig 1A, and of CD11b+ Ly6G+ neutrophils and CD11b- cell was determined within TNF+ cells. Data are shown as mean + SD of 3 individual mice from one representative out of three independent experiments. * p<0.05 compared to other populations (B) Il10 gene expression in liver Ly6C+ monocytes, Ly6C- monocytes and macrophages purified from CX3CR1-GFP+/- mice at day 21 pi was normalized against S12 gene expression and expressed relatively to gene expression in non-fractionated CD11b+ liver cells. Data are shown as mean + SD of 3 individual mice from one representative out of three independent experiments. # p<0.05 compared to non-fractionated CD11b+ myeloid cells; § p<0.05 comparing populations linked by horizontal bar.
Fig 4.
Ly6C- monocytes limit TNF production by Ly6C+ monocytes in T. congolense-infected mice.
(A) Liver Ly6C+ monocytes (Ly6C+ Mo) and MHC-II- to lo Ly6C- monocytes (Ly6C- Mo) purified from CX3CR1-GFP+/- mice at day 7 pi and 21 pi, respectively, were cultured at indicated ratio. TNF concentration in cell supernatants was measured. Data are shown as mean + SD of 1 representative out of five independent experiments. * p<0.05 compared to Ly6C+ monocytes cultured alone. (B-D) Liver MHC-II- to lo Ly6C- monocytes purified from CD45.2 CX3CR1-GFP+/- mice at day 21 pi were transferred in CD45.1 WT mice at day 4 and day 6 pi. Control mice received HBSS. Different parameters were evaluated in recipient mice at d7 pi: (B) Spontaneous TNF levels in liver Ly6C+ monocytes (left panel) and CD11b+ Ly6C- cells (right panel) from control or Ly6C- monocyte-treated mice determined as mean fluorescence intensity difference (dMFI) between anti-TNF and isotype control antibodies. (C) Spontaneous TNF concentration in supernatants of liver non-parenchymal cell cultures and (D) serum alanine aminotransferase (ALT) levels. Data are shown as mean + SD of 3 individual mice of one representative out of three independent experiments. * p<0.05 compared to control mice.
Fig 5.
Depletion of Ly6C- monocytes in Nr4a1-/- mice increases TNF production and liver damage in T. congolense—infected mice.
Different parameters were evaluated in WT and Nr4a1-/- mice at day 21 pi. Liver Ly6C+ monocytes (Ly6C+ Mo), Ly6C- monocytes (Ly6C- Mo) and macrophages (MF) were gated as in S5A Fig in S1 Text. (A) Numbers of Ly6C+ monocytes, Ly6C- monocytes and macrophages within liver non-parenchymal cell population. (B) Spontaneous TNF levels in Ly6C+ monocytes and CD11b+ Ly6C- cells determined as mean fluorescence intensity difference (dMFI) between anti-TNF and isotype control antibodies. (C,D) TNF and (F,G) IL-10 concentration in (C,F) supernatants of liver non-parenchymal cell cultures or (D,G) in blood serum. (E) ALT levels in blood serum. Data are shown as mean + SD of 3 individual mice of one representative out of five independent experiments. *p<0.05 compared to WT mice.
Fig 6.
Ly6C- monocytes through IL-10 production limit TNF production by Ly6C+ monocytes in T. congolense-infected mice.
(A) Liver Ly6C+ monocytes (Ly6C+ Mo) and MHC-II- to lo Ly6C- monocytes (Ly6C- Mo) purified from CX3CR1-GFP+/- mice at day 7 pi and 21 pi, respectively, were cultured alone or together at indicated ratio in presence of control (-) or neutralising anti-IL-10R antibody. TNF concentration in cell supernatants was measured. Data are shown as mean + SD of 1 out of five independent experiments. * p<0.05 compared to Ly6C+ monocytes cultured alone, § p<0.05 comparing populations linked by horizontal bar. (B-D) Liver MHC-II- to lo Ly6C- monocytes gated as described in Fig 5A in S1 Text purified from IL-10wt/wt LysM-cre+/+ or IL-10flox/flox LysM-cre+/+ mice at day 21 pi were transferred in CD45.1 WT mice at day 4 and day 6 pi. Control mice received HBSS. Different parameters were evaluated in recipient mice at day 7 pi: (B) TNF levels in liver Ly6C+ monocytes determined as mean fluorescence intensity difference (dMFI) between anti-TNF and isotype control antibodies, (C) Spontaneous TNF concentration in liver non-parenchymal cell culture supernatants, and (D) ALT levels in blood serum. Data are shown as mean + SD of 3 individual mice of one representative out of three independent experiments. § p<0.05 comparing populations linked by horizontal bar.
Fig 7.
Ly6C- monocytes induce differentiation of Ly6C+ monocytes into macrophages in T. congolense-infected WT mice.
(A) Liver MHC-II- to lo Ly6C- monocytes (Ly6C- Mo) purified from CX3CR1-GFP+/- mice at day 21 pi were transferred in CD45.1 WT mice at day 4 and day 6 pi. Control mice received HBSS. At day 7 pi, percentages of Ly6C+ monocytes (Ly6C+ Mo), Ly6C- monocytes (Ly6C- Mo) and macrophages (MF) within liver non-parenchymal cells were determined in recipient mice. Data are shown as mean + SD of 3 individual mice of one representative out of three independent experiments. *p<0.05 compared to control mice. (B) GFP+ Ly6C+ monocytes purified from CD45.2 LysM-GFP mice at day 7 pi were transferred in CD45.1 WT mice at day 12 pi with or without MHC-II-to lo Ly6C- monocytes gated as described in S5A Fig in S1 Text and purified from CD45.2 WT mice at day 21 pi. After 48 h, liver CD45.2+ GFP+ cells in recipient mice were analyzed for CD11b and Ly6C expression. MHC-II expression was then investigated in Ly6C- CD11b+ cells. FACS profiles are representative of 1 out of 9 mice tested in three independent experiments. Percentages of cells in indicated gates are shown as mean ± SD of 3 individual mice of one representative out of three independent experiments. (C) TNF and (D) IL-10 concentration in supernatants of liver non-parenchymal cell cultures from recipient mice. (E) ALT levels in blood serum from recipient mice. Data are shown as mean + SD of 3 individual mice of one representative out of three independent experiments. *p<0.05 compared to recipient mice receiving only Ly6C+ monocytes.
Fig 8.
Ly6C- monocytes induce differentiation of Ly6C+ monocytes into macrophages in T. congolense-infected Nr4a1-/- mice that lack endogenous Ly6C- monocytes.
(A) GFP+ Ly6C+ monocytes (Ly6C+ Mo) purified from CD45.2 LysM-GFP mice at day 7 pi were transferred in CD45.2 Nr4a1-/- mice at day 12 pi with or without MHC-II-to lo Ly6C- monocytes (Ly6C- Mo) gated as described in Fig 5A in S1 Text and purified from CD45.2 WT mice at day 21 pi. After 48 h, liver GFP+ cells in recipient mice were analyzed for CD11b and F4/80 expression. MHC-II expression was then investigated in CD11b+ F4/80+ cells. FACS profiles are representative of 1 out of 9 mice tested in three independent experiments. Percentages of cells in indicated gates are shown as mean ± SD of 3 individual mice of one representative out of three independent experiments. (B) TNF and (C) IL-10 concentration in supernatants of liver non-parenchymal cell cultures from recipient mice. (D) ALT levels in blood serum from recipient mice. Data are shown as mean + SD of 3 individual mice of one representative out of three independent experiments. *p<0.05 compared to recipient mice receiving only Ly6C+ monocytes.
Fig 9.
Differentiation of Ly6C+ monocytes into macrophages requires a physical contact with Ly6C- monocytes.
(A) Ly6C+ monocytes (Ly6C+ Mo) purified from T. congolense-infected CD45.2 WT mice at day 6 pi were cultured alone or with MHC-II- to lo Ly6C- monocytes (Ly6C- Mo) purified from infected CD45.2 LysM-GFP mice at day 21 pi (1: 1 ratio). Similar cultures were performed in transwell plates. Expression of Ly6C, F4/80, Mertk, CD64 or MHC-II (white line) was investigated 7 days later on GFP- Ly6C+ monocyte-derived cells gated as in S5A Fig in S1 Text (grey line: isotype control). Results are representative of 1 out of 9 mice tested in 3 independent experiments. (B) TNF concentration in cell culture supernatants was measured. Data are shown as mean + SD of 1 representative out of three independent experiments. * p<0.05 compared to Ly6C+ monocytes cultured alone; § p<0.05 comparing populations linked by horizontal bar.
Fig 10.
Overview of the interactions between Ly6C+ monocytes, Ly6C- monocytes and macrophages contributing to trypanotolerance.
TNF-producing Ly6C+ monocytes contribute to liver injury in T. congolense-infected mice. Ly6C- monocytes hamper the pathogenic activity of Ly6C+ monocytes through (1) IL-10 production and (2) cell contact that trigger the differentiation of Ly6C+ monocytes into restorative macrophages. Whether IL-10 possibly produced by monocyte-derived macrophages (dashed lines) contribute to hepatoprotective activity is not elucidated.