Dengue vascular leakage is augmented by mast cell degranulation mediated by immunoglobulin Fcγ receptors

Abstract

Dengue virus (DENV) is the most significant human arboviral pathogen and causes ∼400 million infections in humans each year. In previous work, we observed that mast cells (MC) mediate vascular leakage during DENV infection in mice and that levels of MC activation are correlated with disease severity in human DENV patients (St John et al., 2013b). A major risk factor for developing severe dengue is secondary infection with a heterologous serotype. The dominant theory explaining increased severity during secondary DENV infection is that cross-reactive but non-neutralizing antibodies promote uptake of virus and allow enhanced replication. Here, we define another mechanism, dependent on FcγR-mediated enhanced degranulation responses by MCs. Antibody-dependent mast cell activation constitutes a novel mechanism to explain enhanced vascular leakage during secondary DENV infection.

Keywords: Fc receptor; antibodies; dengue virus; immunology; infectious disease; mast cell; microbiology; mouse; vascular leakage.

Conflict of interest statement

The authors declare that no competing interests exist.

Figures

Figure 1.. DENV-specific IgG enhances MC degranulation and vascular leakage.

(A) MC-degranulation (cell line RBL-2H3) in response to DENV2 is enhanced by dose-dependent increases in the DENV E-specific antibody 4G2. Significance was determined by 2-way ANOVA, where * indicates a significant increase over control, while ** indicates a significant increase over DENV alone without 4G2 antibody. (B) In vivo degranulation of peritoneal MCs was enhanced by DENV compared to isotype controls and 4G2+DENV compared to both controls and DENV infection alone. MC degranulation was determined by flow cytometry to quantitate the heparin-containing granules as an assessment of granularity. Heparin staining of MCs (ckit+) was partitioned into high, mid, low, and negative populations (St John et al., 2011). DENV infection resulted in a significant loss of granulated MCs in the mid partition, and a significant increase in the percentage of cells in the heparin-negative partition (presumably degranulated). This was further accentuated by 4G2 treatment so that approximately 70% of ckit+ cells lacked heparin-staining granules in that group, while both mid and low heparin-staining groups were significantly reduced compared to DENV infection alone. Data were analyzed by 2-way ANOVA with Bonferroni's multiple comparison test to obtain p-values for between group comparisons: *p < 0.05, **p < 0.001, ***p < 0.0001. For each group, n = 3 animals and consistent data were obtained in a second independent trial. (C) Images of MCs were taken by confocal microscopy after cytospinning peritoneal lavage. Slides were stained for MC-heparin to reveal granules and DAPI for nuclei. Control MCs were densely granulated, but degranulation could be observed in samples from DENV-infected animals with and without 4G2 pre-sensitization. More granules appeared extracellular on slides in the 4G2+DENV group. (D) Vascular leakage was measured by obtaining hematocrit values 24 hr and 48 hr after infection with DENV, with and without pre-treatment with 4G2. Significance was determined by 1-way ANOVA with Bonferroni's post-test, where * indicates a significant increase over control, while ** indicates a significant increase over both control and DENV alone. (E) PFU equivalents were measured in mice by real time RT-PCR using RNA isolated from spleens, 24 hr after infection with DENV. Means do not differ significantly by Student's un-paired t-test (p = 0.2073, n = 5). DOI:http://dx.doi.org/10.7554/eLife.05291.002

Figure 2.. Antibody specificity governs IgG-enhanced MC degranulation and vascular leakage.

(A) MC degranulation in response to DENV1–4 is enhanced by cross-reactive antibody 4G2. (B) DENV2-specific antibody 3H5 enhances MC degranulation in response to DENV2 but not DENV1, 3 or 4. For A and B, data were analyzed by 2-way ANOVA with Bonferroni's post-test to determine p-values for group comparisons; * indicates a significant enhancement over control (p < 0.05), † indicates a significant increase over DENV treatment with isotype control (IC) for a given serotype (p < 0.05), and ns designates ‘not significant’. (C) Hematocrit values and (D) Evan's blue dye leakage into tissues were measured 24 hr after DENV1 infection of mice that had been pretreated with IC, 4G2, or 3H5. Hematocrit values and EBD detection values were elevated over baseline for all DENV1-infected mice; however, only the cross-reactive antibody 4G2 increased hematocrit values and Evans blue dye leakage over DENV1 infection with IC antibody. For C and D, significance was determined by 1-way ANOVA with Bonferroni's post-test; p < 0.05. (E) Images show the appearance of mouse livers after saline perfusion. For mice given control injections of saline, the perfusion results in complete flushing of the blood and EBD from the liver. In contrast, bruising consistent with vascular leakage could be observed with DENV1 alone infection, which appears enhanced in mice pre-treated with DENV1 cross-reactive antibody 4G2, but similar in animals pre-treated with non-binding antibody 3H5. DOI:http://dx.doi.org/10.7554/eLife.05291.003

Figure 3.. Antibody-enhanced vascular leakage is MC dependent.

(A) WT and Sash mice were given IC antibody or 3H5 injections 24 hr prior to infection with DENV2. At 24 hr post-infection, blood was obtained for hematocrit analysis. WT mice given DENV2 after either IC or 3H5 treatment had significantly elevated hematocrit values compared to uninfected controls while hematocrit values of Sash mice were not significantly (ns) elevated over controls. EBD in the liver tissue was measured at 24 hr after infection and 30 min after injection with EBD. (B) Images showing the organs in the peritoneal cavity, taken prior to saline perfusion, from necropsy of representative WT and Sash mice with 3H5-antibody-enhanced DENV2 infection showing the organs in the peritoneal cavity. EBD can be visually observed in the gut of WT mice but not in Sash mice. (C) WT mice with and without 3H5 pretreatment before DENV2 infection were treated with the MC stabilizer, Cromolyn. Vascular leakage was measured by EBD perfusion 24 hr post-infection, followed by quantitation in liver tissue. For panels (A–C), significance was determined by 1-way ANOVA with Bonferroni's post-test; * indicates a significant increase over control and ** indicates a significant increase over control as well as the DENV2-infected group that was not pre-treated with 3H5. For (C), δ indicates a significant reduction in vascular leakage after Cromolyn treatment, compared to the untreated group. DOI:http://dx.doi.org/10.7554/eLife.05291.004

Figure 4.. MC FcγRIII-induced degranulation promotes antibody-enhanced vascular leakage.

WT and FcγRIII-KO BMMCs were pre-treated with antibodies 4G2 or 3H5, followed by exposure to (A) DENV2 or (B) DENV1 at an MOI of 1. Both 3H5 and 4G2 enhanced WT BMMC degranulation over DENV2 exposure alone in panel (A), but only 4G2 enhanced BMMC degranulation to DENV1 in panel (B). Both the 3H5- and 4G2-enhanced degranulation responses of BMMCs to DENV2, and the 4G2-enhanced degranulation responses to DENV1 were abrogated in FcγRIII-KO BMMCs. For (A–B), 3H5 or 4G2 pretreatment did not change degranulation levels of FcγRIII-KO BMMCs over DENV exposure alone. All statistical comparisons in (A–B) were performed by 1-way ANOVA with Bonferroni's post-test, *p < 0.05. (C) Degranulation is significantly induced by DENV and UV-DENV in mouse BMMCs. (D) Degranulation responses of WT BMMCs to both DENV and UV-DENV were increased significantly in the presence of antibody 4G2. (E) Antibody 4G2 did not enhance degranulation of FcγRIII-KO BMMCs in response to DENV or UV-DENV. In (D–E), baseline control levels are denoted by gray lines. (F) Both WT and FcγRIII-KO BMMCs showed increased levels of DENV2 replication in the presence of 4G2, compared to control BMMCs but the levels were reduced in 4G2-treated FcγRIII-KO BMMCs compared to WT BMMCs treated with 4G2. All comparisons designated by * were significantly different (p < 0.05), as determined by ANOVA with Tukey's multiple comparison test. (G) This panel depicts the experimental design for BMMC adoptive transfer studies. MC-deficient Sash mice were reconstituted with BMMCs produced from either WT or FcγRIII-KO congenic controls. After maturation, all mice were pre-sensitized with 4G2 antibody prior to challenge with DENV2 (1 × 106 pfu) by i.p. injection (or saline control, uninfected). At 24 hr after injection with saline or DENV2, vascular leakage was measured by (H) hematocrit and (I) EBD detection in the liver after saline perfusion, shown as relative detection, normalized to uninfected controls. (G–I) Mice reconstituted with FcγRIII-KO BMMCs showed reduced vascular leakage compared to mice reconstituted with WT BMMCs, demonstrating the role of MC-expressed FcγRIII in antibody-enhanced vascular leakage. Significance was determined in (H) by 1-way ANOVA with Bonferroni's post-test, and in (I) by un-paired Student's t-test; *p < 0.05. DOI:http://dx.doi.org/10.7554/eLife.05291.005

Figure 5.. Vascular leakage in response to live and inactivated DENV after passive immunization.

(A) A schematic depicting the experimental design of serum-adoptive transfer experiments where post-immune serum from DENV2-infected or naïve mice was adoptively transferred into naïve hosts that were then challenged with 1 × 106 pfu of either DENV1 or DENV2. (B) No significant (ns) differences were detected in the levels of DENV1 infection at 24 hr between groups infected after passive transfer of either control serum or serum from DENV2 post immune (p.i.) mice. Significance was determined by Student's un-paired t-test (p = 0.2905, n = 5). DENV1 RNA levels are displayed normalized to actin and the levels in the control group of mice. (C) Hematocrit analysis showed that vascular leakage was enhanced by either a heterologous challenge with DENV1 (p = 0.0001) or a homologous challenge with DENV2 (p = 0.0005), compared to mice provided control serum from naïve animals. For reference, the dashed line represents the baseline hematocrit value for naïve WT mice. (D) Similarly, mice challenged with UV-inactivated DENV (UV-DENV) showed elevated hematocrit values that were similar to mice given live virus. (E) Serum from control (naïve) mice or DENV2 p.i. mice was adoptively transferred to recipient mice. Levels of serum MCPT1 were measured by ELISA 24 hr after challenge of recipient mice with either DENV2 or UV-DENV2. Passive immunization with DENV2 p.i. serum resulted in elevated concentration of the MC activation biomarker, MCPT1, after challenge. For each panel, p ≤ 0.05; n = 5. (F) Mice adoptively transferred serum from mice immunized against the antigen OVA or control serum were challenged with particulate OVA (pOVA). Antigen alone-treated mice did not have elevated hematocrit levels, but mice given OVA p.i. serum had significantly increased hematocrit levels at 24 hr. (G) The experimental design for adoptive transfer experiments where serum from DENV2 post immune animals was transferred into MC-deficient Sash mice, and Sash mice that had been reconstituted (Sash-R) with either FcγRIII-KO BMMCs or WT BMMCs, followed by a heterologous DENV1 challenge. (H) Mice reconstituted with WT MCs had significantly increased vascular leakage based on hematocrit analysis compared to Sash mice alone (p = 0.002), as did mice reconstituted with FcγRIII-KO BMMCs (p = 0.047); however, mice reconstituted with WT MCs showed a further increase over Sash-R mice repleted with FcγRIII-KO BMMCs (p = 0.003). The dashed line represents the baseline average hematocrit value for naive Sash mice. Statistical comparisons in data-containing panels were performed using Student's unpaired t-test. DOI:http://dx.doi.org/10.7554/eLife.05291.006

Author response image 1.

DENV burden in the spleen of MC-deficient and -repleted mice. Spleens were isolated 24h after infection with DENV and real time RTPCR was performed to quantify the viral burden in this DENV-target organ. In contrast to the significant differences in vascular leakage, viral burden did not differ significantly for the same number of mice (n=5; p=0.13).