Host Inflammatory Response to Mosquito Bites Enhances the Severity of Arbovirus Infection

Abstract

Aedes aegypti mosquitoes are responsible for transmitting many medically important viruses such as those that cause Zika and dengue. The inoculation of viruses into mosquito bite sites is an important and common stage of all mosquito-borne virus infections. We show, using Semliki Forest virus and Bunyamwera virus, that these viruses use this inflammatory niche to aid their replication and dissemination in vivo. Mosquito bites were characterized by an edema that retained virus at the inoculation site and an inflammatory influx of neutrophils that coordinated a localized innate immune program that inadvertently facilitated virus infection by encouraging the entry and infection of virus-permissive myeloid cells. Neutrophil depletion and therapeutic blockade of inflammasome activity suppressed inflammation and abrogated the ability of the bite to promote infection. This study identifies facets of mosquito bite inflammation that are important determinants of the subsequent systemic course and clinical outcome of virus infection.

Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Figures

Graphical abstract

Figure 1

Mosquito Bites Facilitate Virus Retention and Replication at Cutaneous Inoculation Sites, Enhance Viraemia and Systemic Dissemination, and Increase Mortality to SFV Infection (A and B) Mice were infected with 103 PFU of SFV4 either in presence (dark gray bars) or absence (light gray bars) of a mosquito bite. Copy number of SFV RNA (E1 gene) and host 18S was determined by qPCR. Virus titers in the serum were also quantified by plaque assay (n ≥ 7). (C) Mice were infected either in the presence (black line) or absence (grey dotted line) of a mosquito bite with SFV4 (n = 10). (D and E) Mice were infected with 250 PFU SFV6 in presence (dark gray bars) or absence (light gray bars, n = 5) of a mosquito bite. (F) Mice were infected either in the presence (black line) or absence (grey dotted line) of a mosquito bite with SFV6 (n = 10). (G) Mice were infected with 104 PFU BUNV in presence or absence of a mosquito bite (n = 6) and the level of viral RNA and infectious titer quantified at 24 hpi by qPCR and plaque assay. Gene expression of the virally encoded M-segment was used to assay BUNV RNA. (H) Mice were injected with either saline or TLR2 ligand Pam3CSK4 or exposed to mosquito bites and edema determined at 3 hpi (n = 8). (I) Mice were infected with 103 PFU SFV4 constituted in differing volumes and SFV E1 RNA copy number determined by qPCR (n = 8). All column plots show the median value ± interquartile range. Results shown are representative of either two or three experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. See also Figure S1.

Figure 2

Mosquito Bites Induce the Rapid Recruitment of Neutrophils (A) Cutaneous immune responses to bites alone, without concurrent virus infection, were compared to mice infected with SFV4 alone, in the absence of bites. Gene expression was determined using Taqman low-density arrays and hierarchical clustering undertaken to group genes with similar patterns of expression. (B and C) Bite-associated gene expression (B) and SFV4 infection-associated genes (C) at 6 hr were validated by absolute qPCR (n ≥ 6). (D) CXCL2 (n = 6) and IL-1β protein levels (n = 10) were determined by ELISA in skin samples from uninfected and SFV4-infected mosquito bite sites. (E and F) Neutrophil infiltration was determined by histology (E) and flow cytometry (F) at 3 hr after bite or infection with SFV4. (E) Tissue sections stained by H&E. Black arrows indicate typical multi-lobed nuclei of neutrophils. (F) Numbers represent percent of CD11bhiLy6Ghi cells of all live cells (n = 4). (G) Neutrophils were present in high numbers by 90 min after bite/infection, peaking at 180 min. All column plots show the median value ± interquartile range. Results shown are representative of either two or three experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. See also Figures S2–S4.

Figure 3

Mosquito Bites Do Not Subvert the Induction of Antiviral Immune Responses (A) SCID mice were infected with 104 PFU SFV4, either in presence or absence of mosquito bites and the levels of viral RNAs determined (n = 8). (B and C) Wild-type mice were either subjected to four sessions of mosquito biting at 1-week intervals or left unexposed to mosquitoes (bite-naive). Mice were then subjected to mosquito biting and gene transcripts assayed in skin at 6 hr after bite (B) or infected with 104 PFU SFV4 into the bite site and serum viraemia determined at 24 hpi (C) (n ≥ 6). (D and E) Cutaneous type I IFN and ISG gene expression was enhanced by the presence of mosquito bites. Mice (n ≥ 5) were infected with SFV4 either in presence or absence of mosquito bites and the cutaneous expression of transcripts determined by absolute qPCR (D), or fold change determined by TLDA (E). Fold change was calculated by comparison to resting skin and was normalized to the level of SFV E1 copy number. All column plots show the median value ± interquartile range. ∗p < 0.05, ∗∗p < 0.01, ns = not significant.

Figure 4

Mosquito Bite-Infiltrating Neutrophils Express IL-1β, Co-ordinate Innate Immune Gene Expression, and Are Required for the Effective Replication and Dissemination of Virus (A–C) Mice were either depleted of neutrophils by i.p injection of the IA8 antibody or control treated with non-depleting 2A3 control antibody, bitten with mosquitoes, and infected with 104 PFU SFV4 (n ≥ 6). (A) Edema in skin was quantified after i.p. injection of evans blue at 4 hpi. (B) CXCL2 transcripts were determined by qPCR in the skin of mice at 6 hr, n = 12. (C) Skin-bite-associated genes were assayed by qPCR at 6 hpi. Untreated, SFV4-infected mice were included for comparison. (D) IL-6 (n = 26) and CXCL2 (n = 12) transcripts correlated with the number of CXCR2 transcripts in mosquito-bitten skin. (E–G) Skin biopsies at 90 min after bite or SFV4 infection were digested to release cells and stained for flow cytometry. Cells were gated based on their IL-1β staining (E) and back-gated onto a Ly6G/CD11b plot as dark gray dots (F). The percent of IL-1β cells that were Ly6GhiCD11hi at 90 min after bite/infection (n = 6, ND = not detected) (G). (H) Skin virus-associated genes were assayed by qPCR at 6 hpi. Untreated, SFV4-infected mice were included for comparison. (I–K) At 24 hpi, SFV RNA copy number was determined in dLN and inoculation site (skin) (I) and in lymphoid tissues distal to inoculation site (J) by qPCR, n = 7, and infectious virus in the serum determined (K) n = 10. (L) SFV RNA was quantified at 6 hr in the draining popliteal LN to determine whether neutrophil depletion could overcome the initial block imposed by bites on early virus dissemination (n = 7). All column plots show the median value ± interquartile range. Results shown are representative of either two or three experiments. See also Figure S5.

Figure 5

Structurally Unrelated Pro-inflammatory Agents and Mosquito Saliva Promote Virus Infection (A) After s.c. administration of TLR2 ligand Pam3CSK4, alum, or mosquito saliva or after mosquito biting or topical application of the phorbol ester TPA, mice were infected with 104 PFU SFV4 at the same site (n ≥ 5), and the level of viral RNA determined by qPCR and infectious titer determined by plaque assay at 24 hpi. (B and C) Mice were depleted of neutrophils using the IA8 antibody or given 2A3 control antibody, treated with a s.c. injection of Pam3CSK4 (B) or topical application of TPA (C), and then infected with 104 SFV4 at the same cutaneous site. At 24 hpi, SFV RNA copy numbers were determined in the skin (n = 5). (D) After s.c. administration of the TLR2 ligand Pam3CSK4, mice were infected with 104 BUNV, and the level of viral RNA (M-segment) and infectious titer quantified at 24 hpi. All column plots show the median value ± interquartile range. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ns = not significant. See also Figure S6.

Figure 6

Mosquito Bite Enhancement of Virus Infection Is Dependent on IL-1β (A) Numbers of neutrophils infiltrating SFV4-infected bite sites were quantified in WT and Il1r1−/− mice (n = 6). (B and C) WT and IL-1R-null mice (n ≥ 6) were infected with 104 SFV4 at mosquito bite sites or resting skin. SFV RNA copy numbers (B) and viraemia were determined (C) at 24 hpi. (D–G) Mice were treated with the caspase-1 inhibitor Z-YVAD-FMK i.p. (1.5 mg/kg) or vehicle control, bitten by mosquitoes, and then infected with 104 SFV4 at the bite site. (D) Serum IL-1β expression was determined by ELISA (n = 10). (E) Cutaneous bite-associated innate immune transcripts CXCL2 and IL-6 were determined by qPCR at 3 hpi (n = 5). (F and G) Neutrophil influx into bite sites (n ≥ 6) was ascertained by assaying cutaneous CXCR2 expression (F) and the frequency of Ly6GhiCD11b+SSChi cells by flow cytometry at 3 hpi (G). (H–K) Mice were treated with Z-YVAD-FMK i.p. and infected with 104 SFV4 in the absence (H) or presence (I–K) of mosquito bites. SFV RNA was determined by qPCR at 24 hpi (I) and at PID5 (K). Viraemia at 24 hpi was determined by plaque assay (J) (n = 6). (L) Survival curve of mice infected with SFV6 at bite sites with or without treatment with caspase-1 inhibitor Z-YVAD-FMK i.p. at time of infection (n = 15). Results shown are representative of either two or three experiments. All column plots show the median value ± interquartile range. ∗p < 0.05, ∗∗p < 0.01.

Figure 7

CCR2-Dependent Migration and Infection of Myeloid Cells Is Required for Mosquito Bite Enhancement of Virus Infection (A) Mice were infected intradermally with SFV4(Xho)-EGFP (green) in back dorsal skin and at 6 hpi sections stained for lyve1 (red) and DAPI (blue). Arrows indicate double-positive cells in lower magnification image. (B) Skin SFV4(Xho)-EGFP-infected cells were identified by both their expression of EGFP and staining against EGFP and back-gated onto a CD11b/Lyve1 plot, represented as green dots. Luciferase-expressing SFV4 was used as a control. (C) Quantification of CD11b+Ly6Chi myeloid cell numbers in bite sites at 4 and 16 hr. Live cells were first gated for CD45hi cells and to remove Ly6Ghi neutrophils (n = 4). (D) CD11b and Gr-1 staining for all cells (left) and back gating of virus-infected cells at 16 hpi (green dots, right plot). (E) Skin sections were stained for the bone-marrow-derived myeloid marker ER-HR3 (red) in SFV4(Xho)-EGFP-infected skin at 16 hpi. (F) Percentage CD45+Lyve1+ macrophages numbers of all live cells at 24 hpi in the skin (n = 4). (G and H) SFV4-infected bite sites (n = 4) were digested at 16 hpi to release cells and CD11b+ cells sorted on columns to generate a CD11b− fraction and a CD11b+ enriched fraction (G). Infectious virus released by each cell fraction after 6 hr in culture was quantified by plaque assay (H). (I) Gene transcripts for bite-associated innate immune genes and viral RNA at 4 hpi (n ≥ 7). (J) Level of neutrophils at 4 hpi and monocytes at 18 hpi in the skin were determined by flow cytometry (n ≥ 4). (K and L) Ccr2−/− mice are protected from bite-enhanced arbovirus infection at 24 hpi. Wild-type and Ccr2−/− mice were infected with either 104 SFV4 (K) or 104 PFU of the genetically unrelated arbovirus BUNV (L) in the presence or absence of a mosquito bite. (K) qPCR analysis of SFV RNA at 24 hr in the skin and viraemia at 24 hpi (n = 6). (L) qPCR analysis of BUNV M-segment RNA at 24 hr in the inoculation site (skin), remote tissue (spleen), and viraemia at 24 hpi (n = 6). All column plots show the median value ± interquartile range. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ns = not significant. Results shown are representative of either two or three experiments. See also Figure S7.