A prominent role for DC-SIGN+ dendritic cells in initiation and dissemination of measles virus infection in non-human primates

Abstract

Measles virus (MV) is a highly contagious virus that is transmitted by aerosols. During systemic infection, CD150(+) T and B lymphocytes in blood and lymphoid tissues are the main cells infected by pathogenic MV. However, it is unclear which cell types are the primary targets for MV in the lungs and how the virus reaches the lymphoid tissues. In vitro studies have shown that dendritic cell (DC) C-type lectin DC-SIGN captures MV, leading to infection of DCs as well as transmission to lymphocytes. However, evidence of DC-SIGN-mediated transmission in vivo has not been established. Here we identified DC-SIGN(hi) DCs as first target cells in vivo and demonstrate that macaque DC-SIGN functions as an attachment receptor for MV. Notably, DC-SIGN(hi) cells from macaque broncho-alveolar lavage and lymph nodes transmit MV to B lymphocytes, providing in vivo support for an important role for DCs in both initiation and dissemination of MV infection.

Figure 1. Infection of DC-SIGN⁺ cells in the lung.

(A) Infection of the DC-SIGN^hi, DC-SIGN^lo and DC-SIGN^- cells in BAL from MV-infected macaques, determined by detection of EGFP in flow cytometry at day 2–5 d.p.i. Each dot represents an individual animal. Lines indicate geometric means. (B) Macroscopic images from EGFP⁺ lung slices collected 3 d.p.i., cultured for additional 3,5,7 or 10 days. (C) Phenotype of cells migrating from the ex vivo cultured lung slice, collected from supernatant after 5 days of culturing (D) Phenotype of EGFP⁺ cells collected from lung slice medium. (E-F) DC-SIGN expression on lung sections from uninfected macaques (E) or 2.d.p.i. (F) Asterisks indicate DC-SIGN reactivity.

Figure 2. Infection in the tracheo-bronchial lymph nodes.

Infection of the DC-SIGN^hi, DC-SIGN^lo and DC-SIGN^- cells in TBLN from MV-infected macaques, determined by detection of EGFP in flow cytometry from 2–5 d.p.i. Each dot represents an individual animal. Lines indicate geometric means.

Figure 3. DC-SIGN is expressed by DCs and a subset of macrophages in lymph nodes.

(A) TBLN cells were stained for DC-SIGN in combination with DC and macrophage markers and analyzed by flow cytometry. Gray areas show negative controls (DC-SIGN single staining). Percentages of positive cells expressing the markers are annotated in the upper right corner. (B) DC-SIGN expression of CD11c⁺ and CD83⁺ cells in TBLNs. (C-D) Dual immunofluorescence staining of DC-SIGN (green) and MAC387 (red) in lung sections 2 or 3 d.p.i. (C i-iv) and axillary lymphoid tissue 4 d.p.i. (D). Nuclei are stained blue with Hoechst.

Figure 4. Macaque DC-SIGN binds mannose structures including MV and transmits MV to CD150⁺ target cells.

(A) CHO cells were transfected with macDC-SIGN. The mean expression levels of DC-SIGN and CD150 of the parental cell line and transfectants are depicted. Gray areas represent isotype controls. (B) MacDC-SIGN binds mannose and fucose structures on fluorescent beads. Binding was blocked by anti-DC-SIGN (20 ug/ml. (C) CHO transfectants were pre-incubated with anti-DC-SIGN antibodies or isotype control (20 ug/ml) before incubation with FITC-labeled UV-inactivated MV. Binding was measured by flow cytometry. (D) Parental and transfected CHO cells were infected with rMV^KSEGFP (MOI 1) and 48 hours post infection EGFP levels were measured in FACS. (E) Cells were incubated with rMV^KSEGFP (MOI 3). After 3 hours cells were washed and CD150⁺ Raji cells were added to the culture. Infection of Raji cells was determined by measuring EGFP in flow cytometry. Transmission was blocked by pre-incubating CHO macDC-SIGN cells with anti-DC-SIGN (20 ug/ml) or mannan (0.25 mg/ml). All data are representatives for at least 2 independent experiments. Error bars represent standard deviations of duplicates.

Figure 5. DC-SIGN expressed by BAL and lymph node cells enhances MV transmission.

(A) DC-SIGN^hi/HLA-DR⁺ (p1), DC-SIGN^lo/HLA-DR⁺ (p2) and HLA-DR^−/DC-SIGN^- (p3) cells were sorted by FACS from BAL and lymphoid tissues of uninfected animals. Gates and percentages of the subsets are depicted. A representative FACS plot of 3 independent experiments is shown. (B) DC-SIGN on sorted BAL cells binds HIV-1 gp120-coated fluorescent beads. Binding was blocked by mannan (0.25 mg/ml). Representative data of 2 independent experiments are shown. (C) CD150 expression of the 3 subsets from BAL and TBLN (black lines) compared to isotype controls (gray areas). Percentages of CD150⁺ cells are depicted in the histograms. (D) Sorted cells from lymph nodes of an uninfected animal were incubated with fluorescently labeled rMV^KSEGFP or medium control. Binding was measured by flow cytometry and the mean fluorescent intensity (MFI) is depicted. (E) Representative example of 2 indepedent ex vivo infections of BAL cells with rMV^KSEGFP (MOI 3) 24 hours post infection. The left (brightfield) and right (EGFP fluorescent) panel are corresponding pictures. (F) Infection of sorted DC-SIGN subsets with rMV^KSEGFP (MOI 3) was determined by measuring EGFP in FACS. Means and standard deviations of 2 independent experiments are shown. (G-H) Cells were incubated with rMV^KSEGFP (MOI 1) for 3 hours. Then the cells were washed and B cells were added. After 24 hours EGFP expression was measured by flow cytometry. For BAL cells, combined data of 2 independent experiments are shown (G). Cells isolated from lymph nodes were pre-incubated with blocking anti-DC-SIGN antibodies (20 ug/ml) for 30 minutes. ** p<0.01 (H). Bars represent the mean of duplicates.