Protective immune responses against West Nile virus are primed by distinct complement activation pathways

Abstract

West Nile virus (WNV) causes a severe infection of the central nervous system in several vertebrate animals including humans. Prior studies have shown that complement plays a critical role in controlling WNV infection in complement (C) 3(-/-) and complement receptor 1/2(-/-) mice. Here, we dissect the contributions of the individual complement activation pathways to the protection from WNV disease. Genetic deficiencies in C1q, C4, factor B, or factor D all resulted in increased mortality in mice, suggesting that all activation pathways function together to limit WNV spread. In the absence of alternative pathway complement activation, WNV disseminated into the central nervous system at earlier times and was associated with reduced CD8+ T cell responses yet near normal anti-WNV antibody profiles. Animals lacking the classical and lectin pathways had deficits in both B and T cell responses to WNV. Finally, and somewhat surprisingly, C1q was required for productive infection in the spleen but not for development of adaptive immune responses after WNV infection. Our results suggest that individual pathways of complement activation control WNV infection by priming adaptive immune responses through distinct mechanisms.

Figure 1.

Complement is activated in vivo in response to WNV infection. Levels of functional (A) C3 and (B) C4 were determined by erythrocyte hemolysis assay of serum samples from naive and WNV-infected mice. Differences in the C3 and C4 activity between naive and WNV-infected mice were statistically significant (P < 0.05). (C) Serum complement activation was evaluated by Western blot using equal volumes of serum (20 μl of 1/50 dilution) from naive wild-type and C3^−/− mice and WNV-infected (day 2) wild-type mice. Bands corresponding to the C3 α chain (100 kD), C3 β chain (75 kD), and C3dg (38 kD) are labeled.

Figure 2.

All pathways of complement activation are required for survival of WNV infection. Wild-type (n = 95), C1q^−/− (n = 60), C4^−/− (n = 34), fD^−/− (n = 57), fB^−/− (n = 50), and C5aR^−/− (n = 50) C57BL/6 mice were infected with 10² PFU by footpad injection in at least three independent experiments. Significant decreases in survival as compared with wild-type mice were noted for all complement deficient mice (P ≤ 0.0004), except C5aR^−/− mice (P = 0.5).

Figure 3.

WNV infection in serum and lymphoid tissues. WNV RNA levels in the serum (A), spleen (C), and draining inguinal lymph node (D) of wild-type, C1q^−/−, C4^−/−, fD^−/−, and fB^−/− mice at the indicated days were determined by quantitative RT-PCR from at least 4–6 independent mice per time point per group. Infectious WNV burden in the spleen (B) of wild-type, C1q^−/−, C4^−/−, fD^−/−, and fB^−/− mice at the indicated days was determined by plaque assay of samples from 8–15 mice per time point per group. The dotted line indicates the limit of sensitivity of the assay. Asterisks indicate time points at which differences are statistically significant compared with wild type.

Figure 4.

WNV infection in CNS tissues. Infectious WNV burden in (A) spinal cord and (C) brain from wild-type, C1q^−/−, C4^−/−, fD^−/−, and fB^−/− mice was determined by plaque assay of samples from 8–15 mice per time point per group. Scatter plots of (B) spinal cord and (D) brain titers from individual mice at day 4 or 10 are shown to the right of each graph. The limit of sensitivity of the assay and statistical significance are as described in Fig 3.

Figure 5.

WNV antigen staining in the brain. The brains of wild-type, C1q^−/−, C4^−/−, and fD^−/− mice were harvested on day 10, sectioned, and stained for WNV. Representative images of the cerebellum, cortex, and brain stem were determined after staining three to four mice per group. Bar, 200 μm.

Figure 6.

Humoral responses against WNV. Serum samples from wild-type, C1q^−/−, C4^−/−, fD^−/−, and fB^−/− mice were collected at the indicated time points. Titers of specific IgM (A) and IgG (B) against WNV were calculated after incubating serum samples with absorbed control or WNV E proteins. The isotype of WNV-specific IgG responses (C) was analyzed on day 8 after infection. Neutralizing activity (D) of serum samples from wild-type and fB^−/− mice on day 8 after infection was determined by a flow cytometry–based neutralization assay. Data are an average of at least three independent experiments performed in duplicate and reflect 5–10 mice per group. Asterisks indicate time points at which differences are statistically significant compared with wild type (P < 0.05).

Figure 7.

T cell activation and trafficking after WNV infection. (A) Mock or WNV-infected splenocytes from wild-type, C1q^−/−, C4^−/−, fD^−/−, and fB^−/− mice on day 8 were harvested and stimulated ex vivo. Cells were stained for CD4 or CD8 and intracellular IFNγ and analyzed by flow cytometry. Data are an average of at least three independent experiments and reflect 6–10 mice per group. Asterisks indicate differences from mock infected that are statistically significant (*P < 0.05; **P < 0.005). (B) Representative flow cytometry profiles showing intracellular IFNγ staining of splenic CD8⁺ T cells after WNV infection in wild-type or complement-deficient mice. The percentage of double-positive cells is indicated in the top right corner. (C) Brains were harvested from WNV-infected wild-type, C1q^−/−, C4^−/−, and fB^−/− mice on day 9. Leukocytes were isolated by percoll gradient centrifugation and double-stained for CD3 and CD4 or CD8. The total number of brain infiltrating CD4⁺ or CD8⁺ T cells was determined by multiplying the total number of leukocytes from three pooled brains by the percentage of double-positive cells as measured by flow cytometry. Data are an average of at least three independent experiments and reflect at least five groups of three mice. Asterisks indicate differences from wild type that are statistically significant (*P < 0.05; **P < 0.005). (D) Representative flow cytometry profiles showing CD3⁺CD8⁺ T cells in the brain after WNV infection in wild-type or complement-deficient mice.