Figure 1.
CD11c Cre+ Ifnarf/f, LysM Cre+ Ifnarf/f and Ifnar−/− mice are vulnerable to WNV infection.
A. Survival of eight- to ten-week-old mice after inoculation with 102 PFU of WNV by footpad injection. Survival differences were statistically significant between Ifnarf/f and CD11c Cre+ Ifnarf/f (***, P<0.0001), Ifnarf/f and LysM Cre+ Ifnarf/f (***, P<0.0001), and Ifnarf/f and Ifnar−/− mice (***, P<0.0001) but not between Cre− Ifnarf/f (Ifnarf/f) and CD19 Cre+ Ifnarf/f (n = 20, CD11c Cre+ Ifnarf/f, n = 18, LysM Cre+ Ifnarf/fn = 15, Ifnarf/f, n = 15 Ifnar−/−, and n = 8 CD19 Cre+ Ifnarf/f). B. Analysis of IFNAR expression from hematopoietic cells of Ifnar−/−, CD11c Cre+ Ifnarf/f, and Cre− Ifnarf/f infected mice (n = 6 to 8 per group). Blood was harvested 48 hours after WNV infection, and cells were stained with MAbs against IFNAR, CD11c, CD3, CD8, CD4, and CD19. The relative staining of IFNAR on CD11c+CD8+CD3−, CD11c+CD4+CD3− and CD19+CD11c− CD3− is shown. The data is pooled from two independent experiments after normalization of Ifnar expression on Cre− Ifnarf/f cells within a given experiment. C. Survival of eight week-old mice infected with 10 PFU of CHIKV by footpad injection. Survival differences were statistically significant between Ifnarf/f and Ifnar−/− mice (***, P<0.0001) but not between Ifnarf/f and CD11c Cre+ Ifnarf/f or Ifnarf/f and LysM Cre+ Ifnarf/f (n = 8, CD11c Cre+ Ifnarf/f, n = 8 LysM Cre+ Ifnarf/f, n = 12 Ifnarf/f, and n = 12 Ifnar−/− mice). The CD11c-Cre recombinase deletes Ifnar on greater than 95% of conventional CD11chigh dendritic cells and ∼50% of plasmacytoid dendritic cells [88]. The LysM-Cre recombinase deletes Ifnar on mature MØ, granulocytes, and monocytes, with partial gene deletion (∼16%) in CD11c+ splenic dendritic cells [89].
Figure 2.
Viral infection in WNV-infected CD11c Cre+ Ifnarf/f, Cre− Ifnarf/f, and Ifnar−/− mice.
A–G. Viral burden in peripheral and CNS tissues after WNV infection. Eight- to ten-week-old mice were inoculated with 102 PFU of WNV by footpad injection. Levels of infectious virus in the (A) spleen, (B) liver, (C) lung, (D) kidney, (E) brain, (F) heart, and (G) serum were determined from samples harvested 48 hours post-infection using focus-forming assays. Data are shown as FFU per mg of tissue or per ml of serum for six to eight mice per time point. The dotted line represents the limit of sensitivity of the assay and error bars indicate standard deviation (SD). Asterisks indicate values that are statistically significant (***, P<0.0001) compared to Cre− Ifnarf/f mice. H. Blood was harvested 48 hours after WNV infection, and cells were stained with MAbs against CD11c, CD11b, CD3, CD8, CD4, GR-1, and CD19 followed by intracellular staining against the WNV E protein with a combination of two anti-WNV MAbs (WNV E16 and WNV E18). A representative contour plot is provided and shows intracellular WNV antigen levels (red arrows) in cells at 48 hours after inoculation. The percentage of WNV-infected cells for each cell population from each group is shown in the graphs immediately below.
Figure 3.
Tissue analysis of WNV-infected CD11c Cre+ Ifnarf/f mice.
A–C. Histological analysis of the (A) liver, (B) spleen, (C) LN, and (D) brain of Cre−Ifnarf/f (Ifnarf/f), CD11c Cre+ Ifnarf/f, and Ifnar−/− mice. Representative images are shown 48 hours after WNV infection of organs from five to six mice for each group. Inset images show staining at a higher magnification (100×). E–H. TUNEL staining of (E) liver, (F) spleen (G) LN, and (H) brain of Cre−Ifnarf/f (Ifnarf/f), CD11c Cre+ Ifnarf/f, and Ifnar−/− mice 48 hours after WNV inoculation. Cells were counter stained with DAPI. Representative images are shown from five or six mice for each group. I–L. Detection by IHC of WNV antigen in (I) liver, (J) spleen (K) LN, and (L) brain of Cre−Ifnarf/f (Ifnarf/f), CD11c Cre+ Ifnarf/f, and Ifnar−/− mice 48 hours after infection. Representative images are shown of sections from three or four mice for each group. All sections were counterstained with hematoxylin; magnifications are 40×. Insets at higher magnification also are shown.
Figure 4.
Blood chemistry reveals hepatic and renal injury in mice lacking Ifnar expression on myeloid cells.
(A) Blood urea nitrogen (BUN), (B) creatinine (Cr), (C) Alanine aminotransferase (ALT), (D) aspartate aminotransferase (AST), and (E) glucose (GLU) levels were analyzed in the serum of Ifnar−/−, CD11c Cre+ Ifnarf/f, LysM Cre+ Ifnarf/f, and Cre− Ifnarf/f mice (n = 8 to 12 for each group) 48 hours after inoculation with 102 PFU of WNV. Data are pooled from four independent experiments (*, P<0.05; **, P<0.01, ***, P<0.001).
Figure 5.
Serum cytokine levels in WNV-infected mice.
A. Ifnar−/−, CD11c Cre+ Ifnarf/f, LysM Cre+ Ifnarf/f, and Cre− Ifnarf/f mice (n = 13 for each group) were infected 102 PFU of WNV. 48 hours later, serum was collected and the concentration of IL-1ß, IL-6, and TNF-α was determined. Mean values ± SD are shown. B. Forty-eight hours after WNV infection splenic CD11c+ cells were isolated from Ifnar−/−, CD11c Cre+ Ifnarf/f, and Cre− Ifnarf/f mice by positive selection with antibody-coated magnetic beads (n = 3 for each group). Total RNA was prepared and qRT-PCR was used to determine the amount of IL-1ß, IL-6, TNF-α, and WNV E RNA. The mean values ± SD are shown. The data is expressed as fold-increase in mRNA relative to samples from WNV-infected Cre− Ifnarf/f mice.
Table 1.
Cytokine levels in serum of Ifnar−/−, CD11c Cre+ Ifnarf/f, LysM Cre+ Ifnarf/f, and Cre− Ifnarf/f mice during WNV infection.
Figure 6.
Effect of MAVS signaling on cytokine induction.
A–C. WT, Ifnar−/−, Mavs−/−, or Mavs−/−×Ifnar−/− DKO mice (n = 4 for each group) were infected with 102 PFU of WNV and 48 hours later serum was collected. A. Viremia was determined using focus-forming assays. Data are shown as FFU per ml of serum. The dotted line represents the limit of sensitivity of the assay. Error bars indicate the SD. B. At 48 hours after WNV inoculation, splenocytes were gated on CD19−CD11c+CD11blo followed by intracellular staining for WNV antigen. C. The concentration of IL-1ß, IL-6 and TNF-α in serum was determined by cytokine bioplex assay. Mean values ± SD are shown. The IL-1ß, IL-6 and TNF-α cytokine levels were equal between Mavs−/−, Mavs−/−×Ifnar−/− DKO, and WT C57BL/6 control mice. D. A microarray was performed on RNA isolated from spleen of mock-infected mice (n = 2) and WNV-infected WT (n = 3), Mavs−/− (n = 3), Ifnar−/− (n = 3), and Mavs−/−×Ifnar−/− DKO (n = 3) mice. A student's t-test (P≤0.01) was performed to determine the genes that had different expression levels with infection compared to levels in mock infections for each of the four mouse strains (1.5 fold change cut-off). Quantitative analysis of pro-inflammatory cytokines and chemokines is shown on the right. E. WT, Ifnar−/−, Mavs−/−, Ifnar−/−×Mavs−/− DKO BMDCs were infected with WNV and 0, 24 or 48 hours later viral burden in the supernatant was measured. The data are the mean of three independent experiments. Error bars indicate SD. Asterisks denote statistical significance relative to WT cells (*, P<0.05; **, P<0.01). F. Western blot showing phospho-p65 and total (relative) p65 staining in WT, Mavs−/−, Ifnar−/−, Ifnar−/−×Mavs−/− DKO BMDCs at 0 (M, mock), 24 or 48 after WNV infection. ß-actin staining is included as a loading control. The results are representative of two independent experiments.
Table 2.
Cytokine and chemokine levels in serum of WNV-infected WT, Mavs−/−, Ifnar−/−, and Mavs−/−×Ifnar−/− DKO mice.
Figure 7.
TNF-α blockade prolongs survival of WNV infected CD11c Cre+ Ifnarf/f and Ifnar−/− mice.
A. Eight- to ten-week-old CD11c Cre+ Ifnarf/f and Ifnar−/− mice were injected via an intraperitoneal route with 200 µg of anti-TNF-α or isotype control MAbs one day prior to infection with 102 PFU of WNV by footpad injection. Survival differences were statistically significant between anti-TNF-α and isotype control MAb-treated CD11c Cre+ Ifnarf/f mice (***, P<0.0001) and anti-TNF-α and isotype control MAb-treated Ifnar−/− mice (***, P<0.0001). The data is pooled from two independent experiments (n = 8 for all groups). B. Viral burden in serum. Eight- to ten-week-old CD11c Cre+ Ifnarf/f and Ifnar−/− mice were injected with 200 µg of anti-TNF-α or isotype control MAb and Cre− Ifnarf/f were left untreated one day prior to infection with 102 PFU of WNV. Infectious virus in the serum was determined from samples harvested at 48 hours post-infection. Differences were not statistically different. C. Eight- to ten-week-old CD11c Cre+ Ifnarf/f and Ifnar−/− mice were administered 200 µg of anti-TNF-α or isotype control MAb via an intraperitoneal route one day prior to inoculation of 102 PFU of WNV by footpad injection. Glucose (GLU), AST, and ALT levels were analyzed from serum obtained 48 hours after infection. The data are pooled from two independent experiments (n = 8 for all groups). Asterisks indicate differences that are statistically significant (*, P<0.05; **, P<0.01).
Table 3.
Blood chemistry in MAb treated WNV-infected CD11c Cre+ Ifnarf/f and Ifnar−/− mice.
Table 4.
Cytokine levels in serum of Ifnar−/− and CD11c Cre+ Ifnarf/f mice after treatment with anti-TNF-α MAbs and WNV infection.
Figure 8.
Complement activation contributes to liver injury after WNV infection.
A. Microarray analysis of complement genes was performed on RNA isolated from spleen and liver of WNV-infected WT, Mavs−/−, Ifnar−/−, and Mavs−/−×Ifnar−/− DKO mice. Genes that showed statistically significant increases (P<0.05) compared to WT are colored in yellow. Red arrows denote C3 and factor B relative mRNA levels. B–C. Analysis of C3 (left) and factor B (right) levels and split-products (labeled as C3-α2 and Ba) in the serum of Cre− Ifnarf/f, CD11c+ Cre+ Ifnarf/f, and Ifnar−/− mice at 24, 36, and 48 hours after WNV infection (B) as determined by Western blotting. Similar experiments were performed on serum samples at 48 hours after WNV infection from Ifnar−/−, Mavs−/−, and Mavs−/−×Ifnar−/− DKO mice (C). The results are representative of samples from different mice. D. Serum levels of glucose, AST, and ALT in WT, C3−/− and factor B−/− mice after treatment with the IFNAR blocking MAb (MAR1-5A3) and infection with WNV. The serum was harvested 72 hours after infection. For the WT mice, a comparison is made with treatment with the isotype control MAb (GIR-208). The results are the average of two independent experiments with a total of 8 mice per group, and asterisks indicate statistically significant differences (***, P<0.0001). E–F. Viremia and serum cytokines (IL-1ß, IL-6, and TNF-α) at 72 hours in WT, C3−/− and factor B−/− mice after treatment with the IFNAR blocking MAb (MAR1-5A3) and infection with WNV. For the WT mice, a comparison is made with treatment with an isotype control MAb (GIR-208) (*, P<0.05). G. Effect of treatment with C5 blocking MAb on liver injury in WNV-infected CD11c Cre+ Ifnarf/f. CD11c Cre+ Ifnarf/f mice were treated with 1.25 mg (50 mg/kg) of BB5.1 anti-C5 antibody or isotype control (GIR-208) (at days −1 and +2) and infected with 102 PFU of WNV on day 0. At 48 hours, serum was harvested and glucose, ALT, and AST were measured. The results are the mean of two independent experiments with n = 7 or 8 mice in total (**, P<0.01; ***, P<0.001). H. Western blotting analysis of C3 (left) and factor B (right) split-products (labeled as C3-α2 and Ba) in the serum of CD11c+ Cre+ Ifnarf/f and Ifnar−/− mice at 48 hours after WNV infection in animals treated with isotype or anti-TNF-α MAbs. The results are representative of two independent experiments. I. ELISA showing C3a levels in plasma of CD11c+ Cre+ Ifnarf/f and Ifnar−/− mice at 48 hours after WNV infection in animals treated with isotype or anti-TNF-α MAbs. The results are the mean of two independent experiments with a total of n = 6 mice and asterisks indicate significant differences (**, P<0.01).
Figure 9.
Model of sepsis after viral infection of CD11c+ DC or LysM+ myeloid cells.
Myeloid cells that lack signaling (due to Ifnar gene deletion, pharmacological blockade with anti-IFNAR MAbs, or IFN signaling antagonism by viruses) are more susceptible to infection. In the context of WNV infection, increased viral replication results in enhanced RNA PAMP generation, which activates MAVS via recruitment to the mitochondria. This results in downstream activation of IRF-3 and NF-κB and also assembly of the NLRP3 inflammasome. IRF-3 activation and nuclear translocation promotes induction of a limited set of ISGs. NF-κB activation and nuclear translocation promotes expression of pro-inflammatory cytokines (e.g., IL-6 and TNF-α) and chemokines (CCL5 and CXCL10). Soluble TNF-α can modulate endothelial cells function and integrity and also induce complement factor expression in myeloid and hepatic cells, including C3 and factor B. Higher levels of C3 and factor B in the context of increased WNV in plasma results in excessive complement activation (via the alternative pathway), which can liberate the C3a and C5a anaphylatoxins. Along with TNF-α, these promote changes to vascular permeability and tone that result in hypotension and visceral organ (e.g., liver and kidney) damage. Independently, excessive virus infection in restricted tissues (e.g., spleen and LN) can cause organ damage. The pathological effects of this cascade can be mitigated by administration of blocking MAbs to TNF-α or C5.