Immune surveillance by mast cells during dengue infection promotes natural killer (NK) and NKT-cell recruitment and viral clearance

Abstract

A wealth of evidence supports the essential contributions of mast cells (MCs) to immune defense against bacteria and parasites; however, the role of MCs in viral infections has not been defined. We now report that rodent, monkey, and human MCs are able to detect dengue virus (DENV), a lymphotropic, enveloped, single-stranded, positive-sense RNA virus that results in MC activation and degranulation. We observe that the response of MCs to DENV also involves the activation of antiviral intracellular host response pathways, melanoma differentiation-associated gene 5 (MDA5) and retinoic acid inducible gene 1 (RIG-I), and the de novo transcription of cytokines, including TNF-α and IFN-α, and chemokines, such as CCL5, CXCL12, and CX3CL1. This multifaceted response of MCs to DENV is consequential to the containment of DENV in vivo because, after s.c. infection, MC-deficient mice show increased viral burden within draining lymph nodes, which are known to be targeted organs during DENV spread, compared with MC-sufficient mice. This containment of DENV is linked to the MC-driven recruitment of natural killer and natural killer T cells into the infected skin. These findings support expanding the defined role of immunosurveillance by MCs to include viral pathogens.

Fig. 1.

MCs degranulate in response to DENV. (A) DENV1–4 induced degranulation of RBLs. (B) Comparable RBL degranulation to live and UV-killed DENV2. Percentage degranulation in A and B was compared with spontaneous release from unstimulated MCs. *P < 0.05. (C and D) SEM of RBLs. (C) A single granule emerging from the ruffled surface of a RBL after DENV2 treatment. (D) Cell surfaces are visualized without (Upper) and with (Lower) exposure to DENV. Some extracellular granules are false-colored red. (E) Images of mouse footpad sections of control (Left) and DENV2-injected (2 × 10⁵ pfu of DENV were injected s.c.; Right) footpads. MCs in Left are fully granulated and stain metachromatically (purple), whereas MCs in Right are partially degranulated with free purple granules visible in the surrounding tissue. (Magnification: 20×.)

Fig. 2.

Primate MCs degranulate in response to DENV. (A) Images of whole-mounted monkey skin after ex vivo control (Left) or DENV2 injection (5 × 10⁶ pfu; Right). After 1 h, explants were fixed and stained with the MC granule-specific probe, avidin–TRITC. Lower images reveal details of the boxed areas in the Upper images. Control tissue contains MCs staining densely for granules, whereas DENV-treated tissue contains free granules and cells that appear to be hypogranulated. (Magnification: 20×.) (B) Graph quantifies degranulation by human MCs (LAD2). Degranulation is significantly increased in DENV-treated, compared with control, cells. *P = 0.02. (C) Images of LAD2 cells without (Left) and with (Right) exposure to DENV2. Control cells contain several darkly staining granules. After DENV exposure, intracellular granules are not apparent and morphological changes consistent with granule release, e.g., membrane ruffling, are seen. (Magnification: 60×.)

Fig. 3.

MC induction of viral immunity genes after exposure to DENV. (A) RBLs stained with DAPI and for dengue envelope (4G2) and NS3 at 24 h after DENV infection [multiplicity of infection (MOI) = 5]. Control image depicts stained, uninfected cells. (Magnification: 20×.) (B) Enhanced expression of MDA5 and VISA over 24 h coincides with a twofold increase of DENV NS1. ND, not detected. (C and D) DENV enhances expression of cytokines IL-6, TNF, and IFN-α (C) and chemokines CXCL12, CCL5, and CX3CL1 (D). For B–D, expression for host genes was normalized to levels in control RBLs at 1 h. For NS1, values were normalized to the first detected levels, at 6 h. All values were determined by real-time PCR in RBLs with and without DENV treatment (MOI = 1). (E) IFN-α, TNF, and CXCL10 expression by RBLs (Left) or NS1 levels (Right) at 24 h after DENV treatment in cells with control siRNA or siRNA against RIG-I, MDA5, or TLR3. For B–D, * signifies a significant increase and ** indicates a significant decrease compared with control (P < 0.05).

Fig. 4.

Impaired clearance of DENV with MC deficiency. (A) Viral burden in the DLNs of WT, Sash, and Sash-R mice as determined by real-time PCR for DENV NS1, assessed at 24 h after footpad injection of 2 × 10⁵ pfu of DENV2. * signifies a significant increase compared with WT (P = 0.0002; n = 6–8 for each group). (B) Viral replication detected in DLN sections from WT, Sash, and Sash-R mice by staining for NS3 (red) and dsRNA (green). For isotype and uninfected control images and channel-series staining, see Fig. S5.

Fig. 5.

MC-dependent recruitment of NK and NKT cells to site of DENV injection. Relative numbers of NK1.1⁺ CD3⁻ (A) and NK1.1⁺CD3⁺ (B) cells after infection are increased over saline control tissues in WT and Sash-R mice but decreased in Sash mice, resulting in a significantly reduced fold increase for Sash mice compared with MC-sufficient mice (*P < 0.01; n = 4–6). (C) Microscopy of a footpad section 24 h after injection of 2 × 10⁵ pfu of DENV2 shows that MCs (green) are activated in the vicinity of a recruited NK1.1⁺ (red) and CD8⁺ (blue) cell, based on the presence of proximal extracellular granules (boxed in green in C). A larger magnification of the surrounding tissue is included as Fig. S7. (D) Staining for CD31 (blood vessels; red), MC granules (green), and NK1.1⁺ cells (blue) demonstrates that NK1.1⁺ cells are found within the tissue in areas of MC activation in MC-sufficient mice at 24 h after injection of 1 × 10⁵ pfu of DENV2 but not in uninfected (WT control) or DENV-infected Sash mice. (Magnification in C and D: 20× by confocal microscopy.)