MAIT cells promote inflammatory monocyte differentiation into dendritic cells during pulmonary intracellular infection

Abstract

Mucosa-associated invariant T (MAIT) cells are a unique innate T cell subset that is necessary for rapid recruitment of activated CD4+ T cells to the lungs after pulmonary F. tularensis LVS infection. Here, we investigated the mechanisms behind this effect. We provide evidence to show that MAIT cells promote early differentiation of CCR2-dependent monocytes into monocyte-derived DCs (Mo-DCs) in the lungs after F. tularensis LVS pulmonary infection. Adoptive transfer of Mo-DCs to MAIT cell-deficient mice (MR1-/- mice) rescued their defect in the recruitment of activated CD4+ T cells to the lungs. We further demonstrate that MAIT cell-dependent GM-CSF production stimulated monocyte differentiation in vitro, and that in vivo production of GM-CSF was delayed in the lungs of MR1-/- mice. Finally, GM-CSF-deficient mice exhibited a defect in monocyte differentiation into Mo-DCs that was phenotypically similar to MR1-/- mice. Overall, our data demonstrate that MAIT cells promote early pulmonary GM-CSF production, which drives the differentiation of inflammatory monocytes into Mo-DCs. Further, this delayed differentiation of Mo-DCs in MR1-/- mice was responsible for the delayed recruitment of activated CD4+ T cells to the lungs. These findings establish a novel mechanism by which MAIT cells function to promote both innate and adaptive immune responses.

Figures

Figure 1.

Accumulation of CCR2-dependent CD11b+ DCs in the lungs of MR1−/− mice is impaired after F. tularensis i.n. LVS infection. Wild-type C57BL/6, MR1−/−, and CCR2−/− mice were i.n. infected with a sublethal dose of 2 × 102 CFU F. tularensis LVS, and total lung cells were harvested at the indicated time points after infection to evaluate the presence of myeloid cells. (A) Representative flow cytometry dot plots of lung cells on day 4 after infection, demonstrating the gating scheme used to identify CD103+ DCs (gate R1), macrophages (gate R2), and CD11b+ DCs (gate R3). (B) Accumulation of CD103+ DCs, macrophages, and CD11b+ DCs in the lungs of WT mice after i.n. LVS infection. (C) Total numbers of CD11b+ DCs in the lungs of WT, MR1−/−, and CCR2−/− mice on day 4 after i.n. LVS infection. All data are representative of three independent experiments (n = 5 mice per group) and are the mean ± SEM. *, P < 0.01, compared with LVS-infected WT mice at the same time point. Data were analyzed via one-way ANOVA, followed by the Student-Newman-Keuls multiple stepwise comparison.

Figure 2.

Characterization of CD11b+ DCs in the lungs of mice after i.n. F. tularensis LVS infection. Wild-type C57BL/6 mice were infected with a sublethal dose of 2 × 102 CFU F. tularensis i.n. LVS, and total lung cells were harvested on day 4 after infection. (A) Expression of myeloid cell markers by CD11b+ DCs. In the flow cytometry histograms shown, CD11b+ DCs were first gated as indicated in Fig. 1 A, gate R3. Gray histogram shows fluorescence minus one control, and white histogram shows staining by the indicated antibody. Data are representative of two independent experiments (n = 3 mice per group). (B) Quantitation of myeloid marker expression by lung CD11b+ DCs (based on percentage of positive cells). Data are representative of two independent experiments (n = 3 mice per group). (C) Cytospin preparations of CD11b+ DCs purified from the lungs of 10 LVS-infected WT mice on day 4 after infection by fluorescence-activated cell sorting and examined by light microscopy. Bars, 10 µm. Data are representative of two independent experiments. (D) Lung Mo-DCs and CD11b+ DCs are the same population by flow cytometry analysis. Wild-type C57BL/6 mice were infected with a sublethal dose of 2 × 102 CFU F. tularensis i.n. LVS, and total lung cells were harvested on day 4 after infection to evaluate Mo-DCs and CD11b+ DCs. Representative dot plots of lung cells showing the gating scheme for Mo-DCs (gate R1) and CD11b+ DCs (gate R2). Overlay of the Mo-DC gating scheme is shown in red, and the CD11b+ DC gating scheme shown in blue. Data are representative of three independent experiments (n = 5 mice).

Figure 3.

Inflammatory monocyte recruitment to the lungs of MR1−/− mice is not impaired after i.n. F. tularensis LVS infection. Wild-type C57BL/6, MR1−/−, and CCR2−/− mice were i.n. infected with a sublethal dose of 2 × 102 CFU F. tularensis LVS, and (A) total lung cells were harvested on day 4 after infection to evaluate the presence of Ly6Chi CD11b+ inflammatory monocytes. Representative flow cytometry dot plots are shown, as well as enumeration of the total number of Ly6Chi CD11b+ inflammatory monocytes in the lungs of mice. Data are representative of four pooled experiments (n = 3–5 mice per group) and are the mean ± SEM. *, P < 0.01, compared with LVS-infected WT mice at the same time point. (B) Total bone marrow cells were harvested on day 4 after infection to evaluate the presence of Ly6Chi CD11b+ monocytes. Representative flow cytometry dot plots are shown, as well as the percentage of Ly6Chi CD11b+ monocytes in the bone marrow of mice. Data are representative of three independent experiments (n = 5 mice per group) and are the mean ± SEM. *, P < 0.01, compared with LVS-infected WT mice at the same time point. Data were analyzed via one-way ANOVA, followed by the Student-Newman-Keuls multiple stepwise comparison.

Figure 4.

Differentiation of inflammatory monocytes into Mo-DCs is impaired in the lungs of MR1−/− mice after i.n. F. tularensis LVS infection. Wild-type C57BL/6 and MR1−/− mice were infected with a sublethal dose of 2 × 102 CFU F. tularensis i.n. LVS, and (A) total lung cells were harvested at the indicated time points to evaluate the presence of Mo-DCs (Ly6Chi CD11b+ MHCII+ CD11c+ cells). Data are representative of three independent experiments (n = 5 mice per group), and are the mean ± SEM. *, P < 0.05, compared with LVS-infected WT mice at the same time point. (B) Flow cytometry analyses of Mo-DCs in the lungs of WT and MR1−/− mice on day 4 after infection. Representative dot plots are shown. (C) Analysis of CD11c, CD86, and MHCII expression by Ly6Chi CD11b+ cells in the lungs of mice on day 4 after i.n. LVS infection. Data are representative of three independent experiments (n = 5 mice per group), and are the mean ± SEM. *, P < 0.05, compared with LVS-infected WT mice at the same time point. (D) Cytospin preparations of CD11c− and CD11c+ inflammatory monocytes (Ly6Chi CD11b+ cells) purified from the lungs of LVS-infected mice on day 4 after infection by fluorescence-activated cell sorting and examined by light microscopy (top) or confocal microscopy (bottom) using wheat germ agglutinin-Alexa Fluor 594–conjugated membrane stain (red) and Hoechst nuclear stain (blue). Bars, 10 µm. Lung cells were pooled from 10 mice for flow sorting, and data are representative of three independent experiments. (E) WT mice were infected i.n. with LVS-GFP or LVS, and GFP+ cells were evaluated in CD11c− and CD11c+ inflammatory monocytes (Ly6Chi CD11b+ cells) by flow cytometry on day 4 after infection. Representative dot plots are shown. (F) Quantification of the percentage of GFP+ CD11c− and CD11c+ inflammatory monocytes (Ly6Chi CD11b+ cells) in the lungs of mice on day 4 after infection with either LVS-GFP or LVS (negative GFP control). Data are representative of three independent experiments (n = 5 mice per group) and are the mean ± SEM. *, P < 0.05, compared with LVS-infected WT mice at the same time point. Data were analyzed via one-way ANOVA followed by the Student-Newman-Keuls multiple stepwise comparison.

Figure 5.

Adoptive transfer of Mo-DCs to MR1−/− mice rescues their impairment in the accumulation of activated CD4+ T cells in the lungs after i.n. F. tularensis LVS infection. Wild-type C57BL/6 mice were infected with a sublethal dose of 2 × 102 CFU F. tularensis i.n. LVS, and Mo-DCs were purified from the lungs on day 4 after infection by fluorescence-activated cell sorting using the gating scheme shown in Fig. 1 A, gate R3. 106 Mo-DCs were transferred i.v. to MR1−/− mice on day 3 after i.n. LVS infection. Lungs were harvested from WT mice, MR1−/− mice, and MR1−/− adoptive transfer recipients and the total numbers of CD4+ T cells and activated (CD69+ CD44+) CD4+ T cells were quantified by flow cytometry on day 8 after i.n. LVS infection. Data are representative of three independent experiments, and are the mean ± SEM. *, P < 0.05, compared with LVS-infected WT mice. ^, P < 0.05, compared with LVS-infected MR1−/− mice. Data were analyzed via one-way ANOVA, followed by the Student-Newman-Keuls multiple stepwise comparison.

Figure 6.

Bone marrow monocytes from MR1−/− mice do not exhibit a defect in differentiation into MHCII+CD11c+ cells in response to recombinant cytokines in vitro. Monocytes (Ly6Chi CD11b+ cells) were purified from the bone marrow of naive WT and MR1−/− mice by fluorescence-activated cell sorting and cultured for 3 d in the presence of recombinant IFN-γ or GM-CSF (100 ng/ml), then assessed for coexpression of MHCII and CD11c by flow cytometry. Representative flow cytometry dot plots are shown (A). The percentage (B) and total number (C) of MHCII+ CD11c+ monocytes is shown. Bone marrow cells were pooled and sorted from 50 mice; data includes values ± SEM from three replicates, and is representative of three independent experiments. *, P < 0.01, compared with IFN-γ–treated cells. Data were analyzed via one-way ANOVA, followed by the Student-Newman-Keuls multiple stepwise comparison.

Figure 7.

Vα19iTg T cells promote the differentiation of bone marrow monocytes into MHCII+CD11c+ cells in a GM-CSF–dependent manner in vitro. Monocytes (Ly6Chi CD11b+ cells) were purified from the bone marrow of naive WT and MR1−/− mice by fluorescence-activated cell sorting and cultured for 3 d in the presence of MAIT cells and LVS-infected macrophages, then assessed for coexpression of MHCII and CD11c by flow cytometry. Thy1.2+ cells purified from transgenic Vα19iTgCα−/−MR1+/+ mice were the source of MAIT cells. In some cases, as indicated, neutralizing antibodies or isotype control antibodies were added to the cultures at the time of addition of purified MAIT cells. In A, representative flow cytometry dot plots and the number of MHCII+ CD11c+ monocytes are shown for cultures containing WT bone marrow monocytes and LVS-infected WT macrophages in the presence or absence of Vα19iTg Thy1.2+ cells. In B, the number of MHCII+ CD11c+ monocytes are shown for cultures containing WT bone marrow monocytes, LVS-infected WT macrophages, and either Vα19iTg Thy1.2+ cells or naive WT C57BL/6 Thy1.2+ cells. In C, representative flow cytometry dot plots of MHCII+ CD11c+ monocytes are shown for cultures containing MR1−/− bone marrow monocytes, LVS-infected MR1−/− macrophages, and Vα19iTg Thy1.2+ cells. Graph of the numbers of MHCII+ CD11c+ monocytes obtained from MR1−/− cultures as compared with WT cultures (note that A and B are all from the same representative experiment). In D, IFN-γ and GM-CSF in the supernatants of cultures containing WT bone marrow monocytes, LVS-infected WT macrophages, and Vα19iTg Thy1.2+ cells after 3 d are shown. Bone marrow cells were pooled from 50 mice per experiment; data includes values ± SEM from two pooled experiments, each containing two replicate samples, and is representative of five independent experiments. *, P < 0.01, compared with No T cells. ^, P < 0.01, compared with +Vα19i Thy1.2 cells + control Ab. #, P < 0.01, compared with +Vα19i Thy1.2 cells. Data were analyzed via one-way ANOVA followed by the Student-Newman-Keuls multiple stepwise comparison.

Figure 8.

MAIT cells are required for early GM-CSF production in the lungs after i.n. F. tularensis LVS infection. Wild-type C57BL/6 and MR1−/− mice were infected with a sublethal dose of 2 × 102 CFU F. tularensis i.n. LVS, and (A) the levels of GM-CSF and M-CSF were determined in lung homogenates during the first 5 d of infection (n = 5 mice per group). (B) Expression of CD115 and CSF2Rα by inflammatory monocytes (Ly6Chi CD11b+ cells) in the bone marrow and lungs on day 4 after i.n. LVS infection. Red line, fluorescence minus one control; blue line, staining by the indicated antibody. (C) Enumeration of the total number of Ly6Chi CD11b+ inflammatory monocytes in the lungs of WT, MR1−/−, and GM-CSF–depleted WT mice on day 4 after i.n. LVS infection. (D) Representative flow cytometry dot plots of Mo-DC accumulation in the lungs of WT, MR1−/−, and GM-CSF–depleted WT mice on day 4 after i.n. LVS infection. (E) Enumeration of the total number of Mo-DCs in the lungs of WT, MR1−/−, and GM-CSF–depleted WT mice on day 4 after i.n. LVS infection. Data are representative of four independent experiments (n = 5 mice per group), and are the mean ± SEM *, P < 0.01, compared with WT mice. Data were analyzed via one-way ANOVA followed by the Student-Newman-Keuls multiple stepwise comparison.