Transient Depletion of CD169+ Cells Contributes to Impaired Early Protection and Effector CD8+ T Cell Recruitment against Mucosal Respiratory Syncytial Virus Infection

Abstract

Respiratory syncytial virus (RSV) is a major cause of respiratory viral infections in infants and children. Alveolar macrophages (AMs) play a crucial role in combatting airborne pathogens, strongly express CD169, and are localized in the lung alveoli. Therefore, we used CD169-diphtheria toxin receptor (DTR) transgenic mice to explore the roles of CD169⁺ cells in immune responses to mucosal RSV infection. The administration of diphtheria toxin to CD169-DTR mice induced specific AM depletion and reduced the recruitment of Ly6C^hi monocytes. Notably, CD169⁺ cell depletion reduced levels of innate cytokines, such as interferon-β, IL-6, and TNF-α, in bronchoalveolar lavage fluid during RSV infection without affecting the production of proinflammatory chemokines. Moreover, the depletion of CD169⁺ cells increased the recruitment of inflammatory cells to the lung during the early stage of RSV infection, although not during the later stages of RSV infection. Furthermore, the depletion of CD169⁺ cells reduced the recruitment of effector CD8⁺ T cells to the lungs after RSV mucosal infection. Our findings suggest that modulating the number of CD169⁺ cells to enhance immune responses to RSV infection may be useful as a new therapeutic strategy.

Keywords: CD169; CXCL9/10; alveolar macrophage; respiratory syncytial virus; type I interferon.

Figure 1

Diphtheria toxin (DT) administration to CD169-diphtheria toxin receptor (DTR) mice induces alveolar macrophage (AM) depletion and reduces monocyte recruitment. (A) Lung cells isolated from wild-type (WT) and CD169-DTR mice 1 day after DT treatment were stained with the indicated antibodies and analyzed by flow cytometry. AMs, monocytes, neutrophils, eosinophils, plasmacytoid dendritic cells, dendritic cells (DCs), CD11b⁺ DCs, and CD103⁺ DCs were defined by their surface marker expression. The gating strategy is described in Figure S1 in Supplementary Material. (B) Results are shown as dot graphs. Each dot represents an individual mouse (n = 8 in each group). Statistically significant differences are indicated: *P < 0.05, ***P < 0.001, ****P < 0.0001. Data are representative of three independent experiments.

Figure 2

CD169⁺ cell depletion diminishes innate cytokine secretion into bronchoalveolar lavage (BAL) fluid during respiratory syncytial virus (RSV) infection. At the 9 h later of RSV infection, BAL fluid was collected from diphtheria toxin (DT)-treated wild-type (WT) and CD169-diphtheria toxin receptor (DTR) mice. The levels of interferon (IFN)-β (A) and IL-6 and TNF-α (B) were measured by ELISA. Each dot represents an individual mouse (n = 9 in each group). (C) At the 1 day later of RSV infection, the levels of Oas1 and Isg15 mRNA expression in lung tissues from DT-treated WT and CD169-DTR mice were measured using real-time quantitative PCR. Hprt was used as an internal control. (D) At the 9 h later of RSV infection, the mouse chemokines CXCL1, CCL2, CCL3, CCL4, CCL11, and CXCL13 were monitored using a cytometric bead array. Each dot represents an individual mouse (n = 8 in each group). Statistically significant differences between groups are indicated: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data are representative of three independent experiments.

Figure 3

CD169⁺ cell depletion increases the lung recruitment of inflammatory cells during early respiratory syncytial virus (RSV) infection. At the indicated time points during RSV infection, the lungs of diphtheria toxin-treated wild-type (WT) and CD169-diphtheria toxin receptor (DTR) mice were collected, processed, and analyzed by flow cytometry. The numbers of monocytes and neutrophils (A), alveolar macrophages (B), eosinophils (C), and dendritic cells (D) indicate the frequencies in gated cells. Cell number results are shown in the graphs; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Data are representative of two independent experiments. Each dot represents an individual mouse (n = 7 in each group). Statistically significant differences between groups are indicated: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Data are representative of two independent experiments.

Figure 4

CD169⁺ cell depletion does not affect the lung recruitment of inflammatory cells 5 days after respiratory syncytial virus (RSV) infection. Five days after RSV infection, the lungs of diphtheria toxin-treated wild-type (WT) and CD169-diphtheria toxin receptor (DTR) mice were collected, processed, and analyzed by flow cytometry. The numbers of alveolar macrophages (A), monocytes and neutrophils (B), inflammatory monocytes (C), dendritic cells (D), and eosinophils (E) indicate the frequencies in gated cells. Cell number results are shown in the graphs. Each dot represents an individual mouse (n = 7 in each group). Statistically significant differences between groups are indicated: **P < 0.01. Data are representative of two independent experiments.

Figure 5

CD169⁺ cell depletion is dispensable for the adaptive immune response against respiratory syncytial virus (RSV). Seven days after RSV infection, interferon (IFN)-γ production by activated CD4⁺ or CD8⁺ T cells from lungs was measured by intracellular cytokine staining after stimulation with PMA and ionomycin (for CD4⁺ T cells) (A) or H-2Db-restricted M_187–195 peptides (for CD8⁺ T cells) (C). The frequencies and absolute cell numbers of CD44⁺ IFN-γ⁺ CD4⁺ (A,B) and CD8⁺ (C,D) T cells were assessed. Cell number results from RSV-infected mice are shown in the graphs. Each dot represents an individual mouse (n = 7 in each group). Data are representative of two independent experiments.

Figure 6

CD169⁺ cell depletion reduces the recruitment of effector CD8⁺ T cells in lungs after respiratory syncytial virus (RSV) infection. At the indicated time points after RSV infection, bronchoalveolar lavage fluid was collected from diphtheria toxin-treated wild-type (WT) and CD169-diphtheria toxin receptor (DTR) mice. The levels of CXCL9 and CXCL10 in the lavage fluids were measured by ELISA (A). Seven days after RSV infection, the frequencies of CD8⁺ T cells (B) and CD44^hi CD62L^lo CD8⁺ T cells (C) were assessed by flow cytometry. (D) RSV M peptide-specific CD44^hi CD62L^lo CD8⁺ T cells were detected by flow cytometry. (E) Frequencies of CD44^hi CD62L^lo tetramer⁺ CD8⁺ T cells were quantified from flow cytometry data of RSV-infected mice. (F) Five days after infection, RSV viral titers from lung homogenates were measured on HEp-2 cells. Each dot represents an individual mouse (n = 8 in each group). Statistically significant differences between groups are indicated: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Data are representative of two independent experiments.