IL-27 Negatively Regulates Tip-DC Development during Infection

Abstract

Tumor necrosis factor (TNF)/inducible nitric oxide synthase (iNOS)-producing dendritic cells (Tip-DCs) have profound impacts on host immune responses during infections. The mechanisms regulating Tip-DC development remain largely unknown. Here, using a mouse model of infection with African trypanosomes, we show that a deficiency in interleukin-27 receptor (IL-27R) signaling results in escalated intrahepatic accumulation of Ly6C-positive (Ly6C⁺) monocytes and their differentiation into Tip-DCs. Blocking Tip-DC development significantly ameliorates liver injury and increases the survival of infected IL-27R^-/- mice. Mechanistically, Ly6C⁺ monocyte differentiation into pathogenic Tip-DCs in infected IL-27R^-/- mice is driven by a CD4⁺ T cell-interferon gamma (IFN-γ) axis via cell-intrinsic IFN-γ signaling. In parallel, hyperactive IFN-γ signaling induces cell death of Ly6C-negative (Ly6C^-) monocytes in a cell-intrinsic manner, which in turn aggravates the development of pathogenic Tip-DCs due to the loss of the negative regulation of Ly6C^- monocytes on Ly6C⁺ monocyte differentiation into Tip-DCs. Thus, IL-27 inhibits the dual-track exacerbation of Tip-DC development induced by a CD4⁺ T cell-IFN-γ axis. We conclude that IL-27 negatively regulates Tip-DC development by preventing the cell-intrinsic effects of IFN-γ and that the regulation involves CD4⁺ T cells and Ly6C^- monocytes. Targeting IL-27 signaling may manipulate Tip-DC development for therapeutic intervention.IMPORTANCE TNF/iNOS-producing dendritic cells (Tip-DCs) are at the front line as immune effector cells to fight off a broad range of invading microbes. Excessive development of Tip-DCs contributes to tissue destruction. Thus, identifying master regulators of Tip-DC development is fundamental for developing new therapeutic strategies. Here, we identify Tip-DCs as a terminal target of IL-27, which prevents Tip-DC-mediated early mortality during parasitic infections. We demonstrate that IL-27 inhibits Tip-DC development via a dual-track mechanism involving the complex interactions of effector CD4⁺ T cells, Ly6C^- monocytes, and Ly6C⁺ monocytes. These findings delineate an in-depth view of mechanisms of Tip-DC differentiation that may have significant implications for the ongoing development of IL-27-based immunotherapy.

Keywords: African trypanosomes; IFN-γ; IL-27; Ly6C+ monocytes; Ly6C− monocytes; Tip-DCs; host-pathogen interactions; intravital imaging; liver immunity; murine model of African trypanosomiasis; parasites.

FIG 1

Deficiency of IL-27R signaling increases intrahepatic Ly6C⁺ monocytes and their derivative moDCs and Tip-DCs during infections with African trypanosomes. (A to F) IL-27R^−/− and WT mice (n = 3 to 5/group) were infected with T. congolense. Flow cytometry was performed to determine the accumulation and differentiation of intrahepatic Ly6C⁺ monocytes. (A) Representative plots showing the frequency of Ly6C⁺ monocytes on day 10 postinfection. (B) Frequency and absolute number of Ly6C⁺ monocytes. (C) Representative plots showing the frequency of moDCs (within Ly6C⁺ cells) on day 10 postinfection. (D) Frequency and absolute number of moDCs. (E) Representative plots showing the percentage of Tip-DCs (within moDCs) on day 10 postinfection. (F) Frequency and absolute number of Tip-DCs. (G to I) IL-27R^−/− and WT mice (n = 4/group) were infected with T. brucei. The percentages and absolute numbers of Ly6C⁺ monocytes (G), moDCs (H), and Tip-DCs (I) were determined on day 10 postinfection. The frequencies were quantified among CD45⁺ cells unless otherwise specified. Data are expressed as means ± SEM from 3 independent experiments. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 2

Elimination of Tip-DC development prolongs the survival of infected IL-27R^−/− mice. (A and B) IL-27R^−/− and WT mice (n = 3 to 4/group) were infected with T. congolense (A) or T. brucei (B). The plasma levels of MCP-1 and MCP-3 were determined using an ELISA. (C and D) Survival of IL-27R^−/−, CCR2^RFP/RFP IL-27R^−/−, and CCR2^+/RFP IL-27R^−/− mice (n = 5 to 10/group) infected with T. congolense (C) or T. brucei (D). (E and F) Plasma levels of ALT in IL-27R^−/− and CCR2^RFP/RFP IL-27R^−/− mice (n = 3 to 4/group) on day 10 after infection with T. congolense (E) or T. brucei (F). (G) Intravital imaging of the liver in CCR2^+/RFP IL-27R^−/− and CCR2^RFP/RFP IL-27R^−/− mice showing distinct accumulation patterns of RFP⁺ cells (Ly6C⁺ monocytes) on day 7 after infection with T. congolense. (H) Representative plots showing the frequency of Ly6C⁺ monocytes within CD45⁺ cells in the bone marrow, blood, spleen, and liver of IL-27R^−/− and CCR2^RFP/RFP IL-27R^−/− mice on day 7 after infection with T. congolense. (I) Frequencies (within CD45⁺ cells) and absolute numbers of Ly6C⁺ monocytes in the bone marrow, blood, spleen, and liver as well as percentages (within CD45⁺ cells) and absolute numbers of moDCs and Tip-DCs in the liver of IL-27R^−/− and CCR2^RFP/RFP IL-27R^−/− mice (n = 4/group) on days 7 and 10 after infection with T. congolense. Data are expressed as means ± SEM from 2 to 3 independent experiments. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 3

CD4⁺ T cells and IFN-γ promote Tip-DC development in infected IL-27R^−/− mice. (A) Intravital imaging of the infection-induced liver necrotic site in CXCR6^+/GFP mice on day 7 after infection with T. congolense showing colocalization of CXCR6-GFP⁺ cells and CD4⁺ cells. Phycoerythrin (PE)–anti-CD4 and Alexa Fluor 647 (AF647)–anti-CD8 mAbs were intravenously (i.v.) injected into the mice 10 min prior to imaging. Red, CD4⁺ cells; green, CXCR6-GFP⁺ cells; blue, CD8⁺ cells; dashed circles, necrotic site. (B) A series of liver intravital imaging of CCR2^+/RFP CXCR6^+/GFP IL-27R^−/− mice on day 7 postinfection showing cell-cell contacts between CXCR6-GFP⁺ cells and CCR2-RFP⁺ cells (Ly6C⁺ monocytes) at the necrotic site in real time. Arrows indicate a CXCR6-GFP⁺ cell that was initially associated with a CCR2-RFP⁺ cell (Ly6C⁺ monocyte) and was dissociated 60 min later. (C) Intravital imaging showing distinct extents of accumulation of CXCR6-GFP⁺ cells and CCR2-RFP⁺ cells (Ly6C⁺ monocytes) in the liver of CCR2^+/RFP CXCR6^+/GFP mice, CCR2^+/RFP CXCR6^+/GFP IL-27R^−/− mice, and CCR2^+/RFP CXCR6^+/GFP IL-27R^−/− mice treated with anti-CD4 mAb on day 10 after infection with T. congolense. (D to F) IL-27R^−/− mice (n = 3/group) were infected with T congolense and treated with anti-CD4 or anti-IFN-γ mAbs to deplete CD4⁺ T cells or neutralize IFN-γ. Flow cytometry was performed to determine Ly6C⁺ monocytes, moDCs, and Tip-DCs in the liver on days 7 and 10 postinfection. The frequencies (within CD45⁺ cells) and absolute numbers of Ly6C⁺ monocytes (D), moDCs (E), and Tip-DCs (F) are shown. Data are expressed as means ± SEM from 2 to 3 independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 4

Reduced Ly6C⁻ monocytes lead to exacerbated Tip-DC development in infected IL-27R^−/− mice. (A) Intravital imaging showing CX3CR1-GFP⁺ cells accumulated within the clusters of the CCR2-RFP⁺ cells in the liver of CCR2^+/RFP CX3CR1^+/GFP mice. (B to D) IL-27R^−/− and WT mice (n = 3 to 4/group) were infected with T. congolense. Flow cytometry was performed to analyze CD11b⁺ Ly6C⁻ cells (gated on CD45⁺ cells) and Ly6C⁻ monocytes (gated on CD11b⁺ Ly6C⁻ F4/80^lo) in the liver on days 7 and 10 postinfection. (B and C) Representative plots showing the frequencies of CD11b⁺ Ly6C⁻ cells (B) and Ly6C⁻ monocytes (within CD11b⁺ Ly6C⁻ F4/80^lo cells) (C). (D) Percentages and absolute numbers of CD11b⁺ Ly6C⁻ cells and Ly6C⁻ monocytes. (E to H) IL-27R^−/− mice (n = 6/group) were infected with T. congolense and i.v. injected with 2 × 10⁶ Ly6C⁻ monocytes on days 0 and 3 postinfection. (E to G) Flow cytometry was performed to analyze the frequencies and absolute numbers of Ly6C⁺ monocytes (E), moDCs (F), and Tip-DCs (G). The frequencies were quantified among CD45⁺ cells unless otherwise specified. (H) Plasma levels of ALT were measured. Data are expressed as means ± SEM from 2 to 3 independent experiments. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 5

CD4⁺ T cells and IFN-γ drive cell death of Ly6C⁻ monocytes in infected IL-27R^−/− mice. (A to C) IL-27R^−/− and WT mice (n = 3 to 6/group) were infected with T. congolense. Flow cytometry was performed to analyze cell death of intrahepatic Ly6C⁻ monocytes on day 10 postinfection. (A) Representative plots showing cell death of Ly6C⁻ monocytes. (B) Frequencies of live, apoptotic, and necrotic Ly6C⁻ monocytes. (C) Median fluorescence intensity (MFI) of CX3CR1 on Ly6C⁻ monocytes. (D to F) IL-27R^−/− mice (n = 3 to 4/group) were infected with T. congolense and treated with anti-CD4 or anti-IFN-γ mAbs to deplete CD4⁺ T cells or neutralize IFN-γ. Cell death of intrahepatic Ly6C⁻ monocytes was analyzed by flow cytometry. (D) Frequency of live, apoptotic, and necrotic Ly6C⁻ monocytes. (E) MFI of CX3CR1 expressed by Ly6C⁻ monocytes. (F) Frequencies (within CD45⁺ cells or as indicated) and absolute numbers of CD11b⁺ Ly6C⁻ cells and Ly6C⁻ monocytes. Data are expressed as means ± SEM from 2 to 3 independent experiments. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 6

IFN-γ drives differentiation of Ly6C⁺ monocytes and cell death of Ly6C⁻ monocytes in a cell-intrinsic manner. (A to E) Ly6C⁺ monocytes were purified from the bone marrow of naive WT and IFN-γR^−/− mice and labeled with PKH26 and CellVue, respectively. The labeled WT and IFN-γR^−/− Ly6C⁺ monocytes were mixed (at a 1:1 ratio) and transferred to IL-27R^−/− mice (n = 4 to 6/group) on day 7 after infection with T. congolense. Forty-eight hours later, the differentiation of donor Ly6C⁺ monocytes was analyzed by flow cytometry. (A) Schematic of the adoptive-transfer experiment. (B) Representative plots showing the percentages of Ly6C⁺ cells before and after adoptive transfer and their differentiation to matured moDCs (MHCII⁺ CD11c⁺) and Tip-DCs. (C) Frequency of Ly6C⁺ cells and Ly6C⁻ cells among the donor cells. (D) Frequency of matured moDCs (MHCII⁺ CD11c⁺) in Ly6C⁺ cells. (E) Frequency of Tip-DCs in moDCs. (F to H) Ly6C⁻ monocytes were purified from the liver of T. congolense-infected WT and IFN-γR^−/− mice and labeled with PKH26 and CellVue, respectively. The labeled WT and IFN-γR^−/− Ly6C⁻ monocytes were mixed (at 1:1 ratio) and transferred to IL-27R^−/− mice (n = 6/group) on day 7 after infection with T. congolense. The survival of donor cells was analyzed 48 h after transfer by flow cytometry. (F) Schematic of the adoptive-transfer experiment. (G) Representative plots showing the frequencies of live, apoptotic, and necrotic donor Ly6C⁻ monocytes. (H) Frequencies of live, apoptotic, and necrotic donor Ly6C⁻ monocytes. MO, monocytes. Data are expressed as means ± SEM from 2 independent experiments. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001. KO, knockout; p.i., postinfection.