Figure 4. Tick salivary protein IsC1ql3 attenuates the host immune response to B. burgdorferi.

(A) The isolated splenocytes were incubated with 1 μg/mL rIsC1ql3 or BSA and stimulated by lipopolysaccharides (LPS) for 6 h. The cytokine genes expression was then evaluated by qPCR.

(B) IsC1ql3 significantly inhibits CCL3 and IFN-γ gene expression in splenocytes upon LPS stimulation. Each dot represents one biological replicate. Data are represented as mean ± SD. Statistical significance was assessed using a non-parametric Mann-Whitney test (**p < 0.01).

(C) The isolated splenocytes were incubated with B. burgdorferi (1 × 10⁶ cells/mL) alone or mixture with 1 μg/mL rIsC1ql3 for 6 h. IFN-γ gene expression was then evaluated by qPCR. IsC1ql3 significantly inhibits IFN-γ gene expression in splenocytes upon B. burgdorferi infection, but with no effect on CCL3 expression. Each dot represents one biological replicate. Data are represented as mean ± SD. Statistical significance was assessed using a non-parametric Mann-Whitney test (*p < 0.05; ns, p > 0.05).

(D) B. burgdorferi-infected nymphs microinjected with ds IsC1qI3 or ds GFP were fed on clean mice for 72 h to assess cytokine genes expression at the tick bite site. IFN-γ and TNF-α gene expression was significantly upregulated in the absence of tick IsC1ql3. Each dot represents one biological replicate. Data are represented as mean ± SD. Statistical significance was assessed using a non-parametric Mann-Whitney test (*p < 0.05).

(E) Increase in proinflammatory chemokine MIG (CXCL9) was observed in all the IsC1qI3-immunized mice compared with the control mice after B. burgdorferi-infected tick bite. Each dot represents one biological replicate. Data are represented as mean ± SD. Statistical significance was assessed using an unpaired t test (*p < 0.05).

(F) Principal-component analysis (PCA) of transcriptome data from splenocytes stimulated by B. burgdorferi with or without IsC1ql3.

(G) Cluster dendrogram and heatmap of transcriptome data from splenocytes stimulated by spirochetes with or without IsC1ql3. Each column represents biological replicates. The phylogenetic relationships of differentially expressed genes are shown on the right tree. The top tree indicates the cluster relationship of the sequenced samples.

(H) Volcano plot of differentially expressed genes. The genes related to IFN-γ are highlighted in orange. The gene names can be found in Table S1.

(I) GO enrichment analysis of transcriptome data from splenocytes stimulated by B. burgdorferi spirochetes with or without IsC1ql3. The second-level GO terms are shown in the plot, and enrichment analysis was performed using functional annotation tool DAVID.

(J) Mammalian IFN-γ induces a tick Rho-like GTPase, resulting in expression of antimicrobial peptide Dae2.²⁹ IsC1ql3 silencing caused increased IFN-γ level in blood-meal-induced tick Dae2 expression during tick feeding. Each dot represents one biological replicate. Statistical significance was assessed using a non-parametric Mann-Whitney test (*p < 0.05).