Differential infection of mononuclear phagocytes by Francisella tularensis: role of the macrophage mannose receptor

Abstract

Francisella tularensis (Ft) is a Gram-negative bacterium and the causative agent of tularemia. It is well established that this organism replicates inside macrophages, but we are only beginning to understand this interface at the molecular level. Herein, we compared directly the ability of Ft subspecies holarctica live-vaccine strain to infect freshly isolated human peripheral blood monocytes, monocyte-derived macrophages (MDM), and cells of the murine macrophage cell line J774A.1 (J774). We now show that unopsonized bacteria infected human MDM fivefold more efficiently than monocytes or J774 cells in standard media. Moreover, enhanced infection of MDM was mediated, in part, by the macrophage mannose receptor (MR). Forming Ft phagosomes accumulated MR, and infection was inhibited by MR-blocking antibody or soluble mannan but not by the dectin-1 ligand laminarin. Up-regulation of MR in MDM (by exposure to interleukin-4) increased Ft phagocytosis, as did expression of MR in J774 cells. Conversely, opsonized Ft were ingested readily by monocytes and MDM. Medium supplementation with 2.5% fresh autologous serum was sufficient to confer opsonophagocytosis and CD11b accumulated in the membrane at sites of Ft engulfment. Infection of monocytes by opsonized Ft was nearly ablated by complement receptor 3 (CR3) blockade. Conversely, MDM used MR and CD11b/CD18 to ingest opsonized organisms. Altogether, our data demonstrate differential infection of mononuclear phagocytes by Ft and define distinct roles for MR and CR3 in phagocytosis.

Fig. 1

Differential infection of mononuclear phagocytes by Ft LVS. LVS at a MOI of 20:1 (A) or H. pylori at a MOI of 10:1 (B) was added to J774 cells in DMEM + 10% HI-FBS or to monocytes and MDM in RPMI + 10% HI-FBS. Phagocytic indices were scored after 1 h at 37°C. Data are the average ± SEM of three independent experiments performed in triplicate. *, P < 0.05; **, P < 0.01, versus MDM.

Fig. 2

MRs mediate phagocytosis of unopsonized Ft. (A) MDM in RPMI + 10% HI-FBS were preincubated with the indicated concentrations of mannan (man), laminarin (lam), or 10 μg/ml anti-MR antibody prior to addition of Ft at a MOI of 20:1. After 1 h at 37°C, phagocytosis was quantified as in Figure 1. Data indicate the mean ± SEM from at least three independent experiments for each condition and are normalized to the no-antibody control (9±2 Ft/100 MDM). *, P < 0.01, versus control. (B) Confocal sections show enrichment of MR on forming (5 min) LVS phagosomes (arrowheads). (C) J774 and J774-E cells in DMEM + 10% HI-FBS were infected with LVS (MOI 20:1), and phagocytosis was quantified after 1 h at 37°C. Data are the mean ± SD of triplicate samples from a representative experiment. *, P < 0.01. (D) MDM were left untreated or exposed to IL-4 for 48 h to induce alternative activation (Alt. activ.) prior to infection with LVS at a MOI of 20:1 for 60 min. Data are the mean ± SEM from three independent experiments performed in triplicate. *, P < 0.05. (E) MDM and alternatively activated MDM were incubated in RPMI containing 10% HI-FBS in the presence and absence of 0.1 mg/ml mannan prior to addition of Ft at a MOI of 20:1, and phagocytosis was quantified after 1 h at 37°C. Data are the mean ± SD of triplicate samples from one experiment representative of three independent determinations. *, P < 0.05, versus no mannan control.

Fig. 3

Serum complement is required for optimal phagocytosis of LVS. (A and B) Ft LVS was added at a MOI of 20:1 to MDM (A) or monocytes (Monos; B) in RPMI supplemented with 10% HI-FBS or 2.5% fresh AS as indicated. Phagocytosis was quantified after 1 h at 37°C. Data are the average ± SEM from three to six independent experiments performed in triplicate. **, P < 0.01. (C) MDM in RPMI containing 2.5% AS were left untreated (Control) or incubated with 25 μg/ml anti-CD18, anti-CD11b, anti-CD11a, or a mixture of anti-CD18 and anti-CD11b blocking antibody prior to addition of Ft. Phagocytosis was quantified after 1 h at 37°C. Data are the mean ± SEM from three independent experiments performed in triplicate and are normalized to the no-antibody control (42±7 Ft/100 MDM). *, P < 0.02; **, P < 0.01. (D) Effect of 25 μg/ml anti-CD18 and anti-CD11b antibody on Ft infection of freshly isolated blood monocytes in RPMI containing 2.5% AS. Data are the mean ± SEM from three independent experiments performed in triplicate and are normalized to the no-antibody control (44±4 Ft/100 monocytes). **, P < 0.01. (E and F) Human MDM (E) and monocytes (F) were infected with LVS in RPMI + 2.5% AS for 5 min. Confocal sections show enrichment of CD11b on forming phagosomes (arrowheads).

Fig. 4

CD11b/CD18 does not mediate phagocytosis of unopsonized Ft. (A) MDM in RPMI containing 10% HI-FBS were left untreated or preincubated with 25 μg/ml anti-CD18, anti-CD11b, anti-CD11a, or a mixture of anti-CD18 and anti-CD11b antibody prior to addition of Ft at a MOI of 20:1, and phagocytosis was quantified after 1 h at 37°C. In all cases, blocking antibodies were without effect (P>0.05). Data are the mean ± SD of triplicate samples from a representative experiment and are normalized to the no-antibody control (6±3 Ft/100 MDM). Similar data were obtained in two other independent experiments. (B) MDM were infected with LVS in RPMI + 10% HI-FBS for 5 min at 37°C. Confocal sections show that CD11b was not recruited to forming phagosomes containing unopsonized bacteria (arrowheads).

Fig. 5

Contribution of MR to uptake of opsonized Ft. (A) MDM were infected with LVS in RPMI + 2.5% AS for 5 min. Confocal sections show MR on forming LVS phagosomes (arrowheads). (B) MDM in RPMI containing 2.5% AS were left untreated (Control) or preincubated with the indicated concentrations of mannan, laminarin, or 10 μg/ml MR blocking antibody prior to addition of Ft at a MOI of 20:1. After 1 h at 37°C, phagocytosis was quantified. Data indicate the mean ± SEM of at least three experiments performed in triplicate for each condition and are normalized to the no-antibody control (102±32 Ft/100 MDM, n=6). *, P = 0.02, versus control.