SAAG-4 is a novel mosquito salivary protein that programmes host CD4 T cells to express IL-4

Abstract

Mosquitoes represent the most important vector for transmitting pathogens that cause human disease. Central to pathogen transmission is the ability to divert the host immune system away from Th1 and towards Th2 responsiveness. Identification of the mosquito factor(s) critical for programming Th2 responsiveness should therefore lead to strategies to neutralize their function and thus prevent disease transmission. In the current study, we used a TCR transgenic adoptive transfer system to screen gene products present in the saliva of the mosquito Aedes aegypti for their ability to programme CD4 T cells to express the signature Th2 cytokine IL-4. The clone SAAG-4 encodes a secreted protein with a predicted size of 20 kDa whose function has previously been uncharacterized. Notably, SAAG-4 reduced host CD4 T cell expression of the signature Th1 cytokine IFN-gamma while simultaneously increasing expression of IL-4. SAAG-4 is therefore the first identified mosquito factor that can programme Th2 effector CD4 T cell differentiation.

Figure 1

Mosquito bites program CD4 cells responding to associated soluble antigen to develop the capacity to express IL-4. Adoptively transferred Thy1.1⁺ CFSE-labeled clonotypic HA-specific CD4 cells were recovered 4 days following adoptive transfer into Thy1.2⁺ recipients treated i.d. with soluble HA peptide and either spot fed at the site of peptide injection with 2 waves of 5 or 10 Ae. aegypti mosquitoes on day −1 and +1 (1^o infestation), 2 waves of 5 or 10 mosquitoes given at days −15, −13, −1 and +1 (2⁰ infestation) or non-infested (control or C). A, Representative CFSE-dilution histograms with percentage of CFSE-diluted clonotypic CD4 cells shown. B, Representative IFN-γ vs IL-4 intracellular staining plots corresponding to the CFSE-diluted clonotypic CD4 cells in panel A. Quantitative analysis (mean ± SEM) of % CFSE-diluted clonotypic CD4 cells (C) and % of divided clonotypic cells expressing IL-4 (D). N=5 per group and asterisks indicate P< 0.05 compared to the control.

Figure 2

The CD4 cell IL-4 programming activity is contained within mosquito salivary gland extract (SGE). Adoptively transferred CFSE-labeled clonotypic CD4 cells were recovered 4 days following adoptive transfer into recipients treated i.d. with soluble HA peptide and pre-treated 1 h earlier with or without 5 or 10 µg SGE. Representative plots (A) and quantitative analysis (B) of clonotypic CD4 cell proliferation, and representative plots (C) and quantitative analysis (D) of intracellular IFN-γ vs IL-4 expression are presented as in Fig. 1. N=5 per group and asterisks indicate P< 0.05 compared to the control.

Figure 3

Mosquitoes induce IL-4 expression at the skin bite site. Three to four Ae. aegypti were allowed to feed on mouse ear and total RNA was extracted from biopsies at the indicated times. Real-time RT-PCR was performed to measure expression of the indicated cytokine mRNAs. GAPDH mRNA was used as a normalizing standard. RNA extracted from ears of mice not exposed to mosquitoes were considered as naïve and assigned an arbitrary value of 1.0, and changes in mosquito-induced cytokine gene expression are expressed as the ratio between mosquito and naïve samples divided by the number of feeding mosquitoes. The asterisk denotes a statistically significant difference between the means of naïve and experimental groups (P<0.05). N=3 per group.

Figure 4

The Ae. aegypti gene P5F9/SAAG-4 induces IL-4 expression in skin. Plasmids expressing the salivary gland proteins Ag5, 34kDa, P1A3, P1E4 and P5F9/SAAG-4 (10 µg) were injected into mouse ears. A, At 3 and 6 h post-injection, RNA samples were prepared and analyzed for IL-4 expression as in Fig. 3 except that samples treated with empty vector were considered as naïve. B, Expression of IL-2, IL-4, IL-10, IL-12 p40, IFN-γ and TNF-α were performed and analyzed in biopsies of skin injected with the P5F9/SAAG-4 vector. GAPDH mRNA was used as a normalizing standard. The asterisk denotes a statistically significant difference between the means of naïve and experimental groups (P<0.05). N=3 per group.

Figure 5

SAAG-4 programs host CD4 cells to express IL-4. Adoptive transfer recipients were treated with 10 mosquitoes or the indicated plasmids (10 µg). Representative plots (A) and quantitative analysis (B) of clonotypic CD4 cell proliferation, and representative plots (C) and quantitative analysis (D and E) of intracellular IFN-γ vs IL-4 expression are presented as in previous figures. N=8 per group and asterisks indicate P< 0.05 compared to the control.