Rapid differentiation of monocytes into type I IFN-producing myeloid dendritic cells as an antiviral strategy against influenza virus infection

Abstract

Myeloid dendritic cells (mDCs) have long been thought to function as classical APCs for T cell responses. However, we demonstrate that influenza viruses induce rapid differentiation of human monocytes into mDCs. Unlike the classic mDCs, the virus-induced mDCs failed to upregulate DC maturation markers and were unable to induce allogeneic lymphoproliferation. Virus-induced mDCs secreted little, if any, proinflammatory cytokines; however, they secreted a substantial amount of chemoattractants for monocytes (MCP-1 and IP-10). Interestingly, the differentiated mDCs secreted type I IFN and upregulated the expression of IFN-stimulated genes (tetherin, IFITM3, and viperin), as well as cytosolic viral RNA sensors (RIG-I and MDA5). Additionally, culture supernatants from virus-induced mDCs suppressed the replication of virus in vitro. Furthermore, depletion of monocytes in a mouse model of influenza infection caused significant reduction of lung mDC numbers, as well as type I IFN production in the lung. Consequently, increased lung virus titer and higher mortality were observed. Taken together, our results demonstrate that the host responds to influenza virus infection by initiating rapid differentiation of circulating monocytes into IFN-producing mDCs, which contribute to innate antiviral immune responses.

FIGURE 1.

Influenza virus induces rapid differentiation of monocytes into mDCs. (A) Human PBMCs were cultured with Poly(I:C) (10 μg/ml), LPS (1 μg/ml), R848 (5 μg/ml), CpGA (10 μg/ml), or A/Bris/59/07 at an MOI of 1 for 16 h. Cells were then analyzed by flow cytometry to assess the frequencies of various cell types. Total DC populations were defined by a lack of lineage marker expression (CD3⁻CD14⁻CD19⁻CD56⁻) and expression of HLA-DR. (B) The percentages of CD11c⁺CD123^low/−HLA-DR⁺Lin⁻ mDCs and CD11c⁻CD123⁺HLA-DR⁺Lin⁻ pDCs in PBMCs cultured with TLR ligands or A/Bris/59/07, as assessed by flow cytometry. **p < 0.01, versus mock-treated group. (C) The percentages of CD14⁺HLA-DR⁺ monocytes and DCs in PBMCs cultured with LPS or A/Bris/59/07, as assessed by flow cytometry. (D) Monocytes were isolated from fresh PBMCs by FACS sorting and cultured with A/Bris/59/07 or A/Brisbane/10/2007 at an MOI = 1. The percentages of monocytes and mDCs were analyzed by flow cytometry. (E) Expression of α-2,6–linked sialic acids was detected by lectin SNA, and expression of α-2,3–linked sialic acids was detected by MAA I on monocytes. (F) Expression of NS1 by CD11c⁺ mDCs as detected by intracellular flow staining. Data are representative of three independent experiments. *p < 0.05 versus mock-treated group.

FIGURE 2.

Live influenza virus infection is required for monocyte differentiation. (A) The percentages of monocytes, mDCs, and pDCs in PBMCs stimulated with TLR ligands or infected with different strains of IAVs (MOI = 1 for A/X-31, A/WSN/33, A/Bris/59/07, and A/Brisbane/10/2007; MOI = 10 for A/Mexico/4108/2009 and A/California/08/2009), as assessed by flow cytometry. (B) The percentages of monocytes, mDCs, and pDCs in PBMCs cultured with B/Brisbane/60/2008 at an MOI = 1 for 16 h, as assessed by flow cytometry. (C) Flow cytometry analysis of the percentages of monocytes and DCs in PBMCs stimulated by live A/Bris/59/07 virus or UV- or heat-inactivated virus. Numbers indicate the percentages of CD14⁺HLA-DR⁺ monocytes (middle panels) and DCs (right panels). (D) Flow cytometric analysis of the percentage of monocytes in PBMCs cultured with A/Bris/59/07 for 6 or 16 h. Data are representative of three independent experiments. *p < 0.05, **p < 0.01 versus mock-treated group.

FIGURE 3.

Influenza virus-induced mDCs exhibit impaired ability to mature and stimulate alloreactive T cells. (A) PBMCs were cultured with TLR ligands and different strains of IAV for 16 h, as in Fig. 2A. The mean fluorescent intensities of HLA-DR, CD86, and CD40 of mDCs were assessed by flow cytometry. (B) Lin⁻HLA⁻DR⁺CD11c⁺CD123^low/− mDCs from LPS-activated or A/Bris/59/07 virus- or mock-infected PBMCs were purified by FACS sorting. The mRNA expression of inflammatory cytokines (IL-12p35, IL-12p40, TNF-α, and IL-6) in mDCs was assessed by real-time RT-PCR using primers listed in Table I. (C) FACS-sorted mDCs were cultured overnight in complete RPMI medium, and the production of IL-12p70, TNF-α, IL-6, RANTES, and IL-8 was examined by Bio-Plex assay. (D) Isolated mDCs were cultured overnight in complete RPMI medium, and the production of MCP-1 and IP-10 was examined by Bio-Plex assay. (E) PBMCs were infected with A/Bris/59/07 for 16 h. Lin⁻HLA⁻DR⁺CD11c⁺CD123^low/− mDCs were purified by FACS sorting. A total of 2 × 10⁴ isolated mDCs was cocultured with 1 × 10⁵ CFSE-labeled naive alloreactive CD45RA⁺CD45RO⁻ CD4 or CD45RA⁺ CD27⁺ CD8 T cells for 5 d. T cell proliferation was assessed by CFSE dilution. Data are representative of three independent experiments. **p < 0.01, ***p < 0.001 versus mock-infected group.

FIGURE 4.

Influenza virus-induced mDCs exhibit antiviral activity. (A) Lin⁻HLA⁻DR⁺CD11c⁺CD123^low/− mDCs in LPS-activated or mock- or A/Bris/59/07 virus-infected PBMCs were purified by FACS sorting. The mRNA expression of IFN-α and IFN-β was examined by real-time RT-PCR. ***p < 0.001, versus mock-treated group. (B) Isolated mDCs were cultured overnight in complete RPMI medium, and the production of IFN-α and IFN-β in the supernatant was assayed using Bio-Plex. Assay range: 12.5–500 pg/ml. (C) The mRNA expression of tetherin, viperin, IFITM3, RIG-I, and MDA5 in purified mDCs was examined by real-time RT-PCR using primers listed in Table I. *p < 0.05, **p < 0.01, ***p < 0.001, versus mock-treated group. (D) A549 cells were preincubated for 30 min with UV-inactivated supernatants from mock- or A/Bris/59/07 virus-infected monocytes; A549 cells were then infected with A/Bris/59/07 for 16 h. Cell lysates were analyzed for influenza virus NP expression by immunoblot. (E) A549 cells were preincubated for 30 min with UV-inactivated supernatants from mock- or A/Bris/59/07 virus-infected monocytes, or A549 cells were pretreated with mock supernatant plus recombinant type I IFN (50 pg/ml or 100 ng/ml) or A/Bris/59/07 supernatant plus 10 μg/ml each of anti–IFN-α and anti–IFN-β Abs. A549 cells were then infected with A/Bris/59/07 for 16 h, and supernatants were collected to measure virus titer by an MDCK cell-based plaque assay. Data are representative of three independent experiments. *p < 0.05 versus mock-treated group, **p < 0.01 versus A/Bris/59/07 supernatant-treated group. ND, Undetectable level of cytokine.

FIGURE 5.

Influenza virus infection accumulates monocytes and mDCs in vivo. (A) Total number of monocytes in lung tissue from influenza virus-infected (PR8, 100 MID₅₀) C57BL/6 (n = 3–6/group) mice was examined by flow cytometry on days 1, 3, 5, 7, and 10 p.i.*p < 0.05, **p < 0.01, versus day 0. (B) Total number of different subsets of DCs in lung tissue from influenza virus-infected (PR8, 100 MID₅₀) C57BL/6 (n = 3–6/group) mice were examined by flow cytometry on days 1, 3, 5, 7, and 10 p.i. *p < 0.05, ***p < 0.001, versus day 0. (C) Expression of NS1 by CD11c⁺ mDCs in lung tissues 5 d post-PR8 infection, as detected by intracellular flow staining. ***p < 0.001, versus mock-treated group. (D) CD11b⁺CD11c⁺ mDCs were isolated by FACS sorting from lung tissue on day 5 p.i. The mRNA expression of IFN-α was examined by real-time RT-PCR. *p < 0.05, versus mock-treated group. (E) Isolated lung CD11b⁺CD11c⁺ mDCs were cultured overnight in complete RPMI medium, and the production of IFN-α in culture supernatants was detected by ELISA. Data are representative of three independent experiments.

FIGURE 6.

Monocyte depletion results in increased mortality and lung viral titer in influenza virus -infected mice. (A) Monocyte-depleted or control C57BL/6 mice (n = 3–6/group) were intranasally infected with PR8 (100 MID₅₀) and sacrificed on day 5 p.i. to examine the total number of monocytes and DCs in lung tissues by flow cytometry. *p < 0.05, **p < 0.01, versus mock-treated group. (B) Survival kinetics of monocyte-depleted or control C57BL/6 mice (n = 5/group) inoculated with PR8 virus. (C) Lung tissue homogenates from monocyte-depleted or control mice (n = 3/group) were analyzed for type I IFN secretion by ELISA. *p < 0.05, **p < 0.01, versus PBS liposome group. (D) Lung tissue homogenates from monocyte-depleted or control mice (n = 3/group) were assayed for virus titer on days 3 and 5 p.i. Data are representative of three independent experiments. *p < 0.05 versus PBS liposome group.