An IRF-3-, IRF-5-, and IRF-7-Independent Pathway of Dengue Viral Resistance Utilizes IRF-1 to Stimulate Type I and II Interferon Responses

Abstract

Interferon-regulatory factors (IRFs) are a family of transcription factors (TFs) that translate viral recognition into antiviral responses, including type I interferon (IFN) production. Dengue virus (DENV) and other clinically important flaviviruses are suppressed by type I IFN. While mice lacking the type I IFN receptor (Ifnar1^-/-) succumb to DENV infection, we found that mice deficient in three transcription factors controlling type I IFN production (Irf3^-/-Irf5^-/-Irf7^-/- triple knockout [TKO]) survive DENV challenge. DENV infection of TKO mice resulted in minimal type I IFN production but a robust type II IFN (IFN-γ) response. Using loss-of-function approaches for various molecules, we demonstrate that the IRF-3-, IRF-5-, IRF-7-independent pathway predominantly utilizes IFN-γ and, to a lesser degree, type I IFNs. This pathway signals via IRF-1 to stimulate interleukin-12 (IL-12) production and IFN-γ response. These results reveal a key antiviral role for IRF-1 by activating both type I and II IFN responses during DENV infection.

Keywords: IFNs; IL-12; IRF-1; IRFs; dengue; flavivirus; innate immunity; interferon; macrophages; mouse models.

Figure 1. Lethality and clinical disease manifestations after DENV infection

WT, DKO, TKO, and Ifnar1^−/− mice were infected with 5 × 10⁶ FFU of DENV2 strain 221 with antibody dependent enhancement (ADE), and (a) lethality, (b) percent weight loss and (c) clinical disease scores were assessed daily for 10 days. Survival was compared between all groups and Ifnar1^−/− mice showed less survival than all other groups tested (log-rank test, P < 0.0001). Data are expressed as mean percent weight loss and clinical scores that were compared daily by one-way analysis of variance (ANOVA) or non-parametric (Kruskal-Wallis) ANOVA with multiple correction, respectively. Levels of viral RNA in the (d) serum (e) spleen, (f) kidney, and (g) liver at 24h and 72h p.i. were determined by quantitative RT-PCR. Data are presented as mean log₁₀ viral GE per mL of serum or per copy of 18S ribosomal RNA of tissues for six to eight mice from two independent experiments. The lower limit of detection is denoted by a dotted line. Viral titers between the gene-deficient and WT mice were compared by ANOVA with Tukey’s multiple comparisons tests. Asterisks indicate differences that are statistically significant (****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05).

Figure 2. Bone marrow derived macrophage (BMDM) response to DENV infection

BMDMs from (a) WT, TKO, and Ifnar1^−/− or (b) DKO and TKO were infected with DENV2 S221 under ADE conditions (MOI of 0.5) and viral replication was measured by focus-forming assay (FFA). Data represents the mean ± standard error mean (SEM) of three independent experiments. The dotted line represents the limit of detection of the assay. (c) BMDMs from DKO and TKO were infected with DENV2 S221 under ADE conditions (MOI of 0.5), RNA was isolated at 24 hours after infection, and relative expression of the indicated target genes was measured by qRT-PCR and normalized to Rplp0. Data represent the average of three independent experiments (black circles) and are expressed as the mean ± SEM. (d) TKO BMDMs were infected as above (panels 2a and 2b) ± IFNAR-blocking Ab (clone MAR1-5A3) added 12hrs prior to infection and viral replication measured by FFA. Data represents the mean ± standard error mean (SEM) of two independent experiments. Log₁₀ viral FFU from gene-deficient, IFNAR-blocking Ab treated, or WT BMDMs were compared to TKO using 2-way ANOVA with correction for multiple comparisons. Relative gene expression (c) was compared using 1-way ANOVA with Tukey’s correction for multiple comparisons. Asterisks indicated differences that were statistically significant (****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05).

Figure 3. Gene expression analysis of splenocytes during DENV infection

(a–b) Chord diagram of significantly upregulated genes after DENV infection determined by RNA-Seq analysis of total splenocytes and their overlap in WT, Ifnar1^−/−, DKO and TKO at (a) 6h and (b) 24h p.i. (c) K-means cluster analysis of differentially expressed genes in WT, Ifnar1^−/−, DKO and TKO splenocytes at baseline and 6h and 24h p.i. (d) GO and KEGG pathway terms significantly enriched in genes contained in Clusters 1–3. Log Benjamini-corrected P-values are shown. (e) Examples of genes contained within Clusters 1–3. Known IRF-1 target genes are highlighted in blue. (f) UCSC genome browser images of Ifng gene from indicated genotypes at 24h p.i. (g) Motifs enriched in the promoters of genes found in Cluster 1 or Cluster 3. (h) WT and TKO mice were infected with 5 × 10⁶ FFU of DENV2 strain 221 with ADE and RNA was isolated at 24 hours after infection from total splenocytes. Relative expression of the indicated target genes was measured by qRT-PCR and normalized to Rplp0. Data represent the average expression level in 6 mock-treated WT, 10 DENV-infected WT, 6 mock-treated TKO and 6 DENV-infected TKO spleens expressed as the mean ± SEM. Relative gene expression was compared using 1-way ANOVA with Tukey’s correction for multiple comparison. Asterisks indicated differences that were statistically significant (****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05).

Figure 4. Effects of blocking IL-12p40 or type I or II IFN signaling on TKO resistance to DENV infection

TKO mice were infected with 5 × 10⁶ FFU of DENV2 strain 221 with ADE and treated with IL-12p40 blocking or isotype control antibodies and (a) lethality, (b) percent weight loss (c) and clinical scores assessed daily for 10 days. (d–i) TKO and Ifnar1^−/− mice were infected with 5 × 10⁶ FFU of DENV2 strain 221 and treated with IFNγ and/or IFNAR blocking or isotype control MAbs. (d, g) Lethality, (e, h) percent weight loss (f, i) and clinical disease scores assessed daily for 10 days. Survival was compared between all groups by the log-rank test and significance between groups is indicated by asterisks. Data are expressed as mean percent weight loss and clinical scores that were compared daily by Student’s t-test with Holm-Sidak correction (b, c) for multiple comparison or one-way analysis of variance (ANOVA) (e, h) or non-parametric (Kruskal-Wallis) ANOVA with multiple correction (f, i) Asterisks indicate statistically significant differences (****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05).

Figure 5. Role of IRF-1 in the host response to DENV infection in vivo and in macrophages

(a–d) BMDMs from indicated genotypes were infected with DENV2 S221 under ADE conditions (MOI = 0.5) and viral replication measured by (a) FFA and (b–c) relative gene expression measured by qRT-PCR. Data represents the mean ± standard deviation (SD) in one representative experiment performed in triplicate and repeated three times. The dotted line represents the limit of detection of the assay. (b–d) Gene expression levels were normalized to Rplp0 and displayed as the fold increase compared to untreated BMDMs. Log₁₀ viral FFU from gene-deficient and WT macrophages were compared by 2-way ANOVA with Tukey’s multiple comparisons tests. Gene expression studies were compared by 1-way ANOVA with correction for multiple comparison. (e–g) TKO, QKO, and Ifnar1^−/− mice were infected with 5 × 10⁶ FFU of DENV2 strain 221 with ADE and (e) lethality, (f) percent weight loss and (g) clinical disease scores were assessed daily for 10 days. Survival was compared between all groups by the log-rank test and significance between groups indicated by asterisks. (f and g) Data are expressed as mean percent weight loss and clinical scores, respectively, that were compared daily by ANOVA (f) and non-parametric (Kruskal-Wallis) ANOVA (g) with multiple correction. WT (n = 4 (8h), 14 (24h), 5 (48h)); TKO (n = 5 (8h), 10 (24h), 5 (48h)); and QKO (n = 4 (8h), 6 (24h), 5 (48h)) mice were infected as above and euthanized at indicated time points for serum collection. Serum levels of IFNβ, IL-12p70 and IFNγ were measured by ELISA. Data are expressed as the mean ± SEM and were compared at each time point by ANOVA with Tukey’s correction for multiple comparisons. Asterisks indicate significant differences (****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05).