Signal transducer and activator of transcription 3 (Stat3) regulates host defense and protects mice against herpes simplex virus-1 (HSV-1) infection

Abstract

Signal transducer and activator of transcription 3 (STAT3) mediates cellular responses to multiple cytokines, governs gene expression, and regulates the development and activation of immune cells. STAT3 also modulates reactivation of latent herpes simplex virus-1 (HSV-1) in ganglia. However, it is unclear how STAT3 regulates the innate immune response during the early phase of HSV-1 lytic infection. Many cell types critical for the innate immunity are derived from the myeloid lineage. Therefore, in this study, we used myeloid-specific Stat3 knockout mice to investigate the role of STAT3 in the innate immune response against HSV-1. Our results demonstrate that Stat3 knockout bone marrow-derived macrophages (BMMs) expressed decreased levels of interferon-α (IFN-α) and interferon-stimulated genes (ISGs) upon HSV-1 infection. In vivo, knockout mice were more susceptible to HSV-1, as marked by higher viral loads and more significant weight loss. Splenic expression of IFN-α and ISGs was reduced in the absence of STAT3, indicating that STAT3 is required for optimal type I interferon response to HSV-1. Expression of TNF-α and IL-12, cytokines that have been shown to limit HSV-1 replication and pathogenesis, was also significantly lower in knockout mice. Interestingly, Stat3 knockout mice failed to expand the CD8⁺ conventional DC (cDC) population upon HSV-1 infection, and this was accompanied by impaired NK and CD8 T cell activation. Collectively, our data demonstrate that myeloid-specific Stat3 deletion causes defects in multiple aspects of the immune system and that STAT3 has a protective role at the early stage of systemic HSV-1 infection.

Keywords: dendritic cells; innate immunity; natural killer cells; type I interferons; viral infection.

Figure 1.. STAT3 KO BMMs show attenuated type I IFN response to HSV-1 infection.

WT or Stat3 KO BMMs were infected with HSV-1 at an MOI of 3. (A) Viral titer in the supernatants at 12 and 24 h after infection was determined by plaque assay on Vero cells with 3 technical replicates. (B) Western blotting of lysates from HSV-1–infected BMMs at the indicated times. (C–M) Gene expression of HSV-1–infected BMMs was determined by quantitative real-time PCR with 3 technical replicates for each sample. All values were normalized to Gusb and compared with the expression of WT BMMs at 0 h, except for IFN-α, which is shown as its relative expression to Gusb. All values are means with 95% confidence intervals. Statistical significance was determined by Student’s t test. *P < 0.05, **P < 0.01, n.d., not determined (below detection threshold).

Figure 2.. Myeloid Stat3 KO mice are more susceptible to HSV-1 infection.

(A) Sex- and age-matched WT or KO mice (n = 5 each) were i.v. injected with 2 × 10⁸ PFU of HSV-1. Survival of infected mice was monitored 3 times daily. Statistical analysis was performed by the log-rank test. (B) Sex- and age-matched WT or KO mice (n = 10 each) were i.v. injected with 1 × 10⁷ PFU of HSV-1. Survival of infected mice was monitored and analyzed as described in panel A. (C) Sex- and age-matched WT or KO mice were i.v. injected with 1 × 10⁷ PFU of HSV-1. Each mouse was weighted immediately before infection and twice daily after infection. All measurements were normalized to the starting weight of each mouse and are expressed as percentages of the starting weight. Statistical analysis was performed by repeated-measure ANOVA using PRISM (GraphPad Software, La Jolla, CA, USA). (D–E) Mice of each genotype (n = 5 each) were infected as described in panel C and were sacrificed 2 dpi. HSV-1 viral loads in the brain or the spleen were determined by quantitative real-time PCR with 3 technical replicates for each mouse. Data are shown as HSV-1 copy number relative to mouse Actb expression. Statistical significance was determined by Student’s t test. **P < 0.01.

Figure 3.. Susceptibility of Stat3 KO mice is not caused by immunopathology in the brain.

WT or KO mice were i.v. injected with 1 × 10⁷ PFU of HSV-1 as described in Fig. 3. Mice of each genotype (n = 5 each) were sacrificed 2 dpi to collect brain tissue. (A–E) Relative expression of iNOS, TNF-α, IFN-α, IFN-β, and IL-12 in the brain was determined by quantitative real-time PCR with 3 technical replicates for each mouse. All values were normalized to Gusb expression and are shown as means with 95% confidence intervals. Statistical analysis was performed with the Student’s t test. n.s., not significant. (F) Brain hemispheres from WT or KO mice sacrificed at 2 dpi were fixed and stained with STAT3, CD45, or Ly6G Abs, as described in Materials and Methods. Microglia showed low levels of CD45 staining. Representative figures from each genotype are shown. (G and H) Relative expression of CCL2 and CXCL2 in the brain was determined by quantitative real-time PCR, as mentioned above.

Figure 4.. Stat3 KO mice express reduced levels of antiviral cytokines in the spleen.

WT or KO mice were infected as described in the caption to Fig. 3. Mice of each genotype (n = 5) were sacrificed 2 dpi to collect spleen tissue. (A–H) Expression of IFN-α, USP18, IFN-β, IFN-γ, iNOS, TNF-α, and IL-12 in the spleen was determined by quantitative real-time PCR with 3 technical replicates for each mouse. All values were normalized to Gusb expression and are shown as means with 95% confidence intervals. Statistical analysis was performed with a t test. **P < 0.01, *P < 0.05, n.s., not significant.

Figure 5.. Stat3 KO mice have decreased CD8⁺ cDC frequency during HSV-1 infection.

Sex- and age-matched WT or KO mice were infected with 1 × 10⁷ PFU of HSV-1 by tail-vein injection. Mock-infected WT (n = 3) and KO mice (n = 3) and infected WT (n = 5) and KO mice (n = 4) were sacrificed 2 dpi, and splenic cells were collected for flow cytometry analysis. (A) Splenic cells were gated on CD11c⁺, followed by CD8, CD11b, and B220 analyses to determine the percentage of CD8⁺ cDC (CD11c⁺CD8⁺CD11b⁻), CD11b⁺ cDC (CD11c⁺CD8⁻CD11b⁺), and pDC (CD11c⁺CD8⁻CD11b⁻B220⁺) populations. Representative figures from 1 mouse of each group are shown. (B–E) Results summarize the percentages of total DCs, CD11b⁺ cDCs, CD8⁺ cDCs, and pDCs from flow analysis. Data shown are means ± sd. Statistical analysis was performed with a Student’s t test. n.s., not significant; *P < 0.05; **P < 0.01.

Figure 6.. Stat3 KO mice have impaired NK and T cell activation during HSV-1 infection.

WT and KO mice were infected and sacrificed for flow cytometry analysis as described in the caption to Fig. 5. (A) Representative figures of 1 mouse from each group are shown. NK cells in the spleen were defined with a CD3⁻NK1.1⁺ gate. Activated NK cells were further gated by CD69⁺ staining. (B and C) Results summarize the percentages of total NK and activated NK cells. (D) Results summarize the percentages of activated CD8 T cells (CD3⁺CD8⁺CD69⁺) in the spleen. (E) The mean fluorescence intensity (MFI) of CD69 was calculated from activated CD8 T cells defined in panel D. Data shown are means ± sd. Statistical analysis was performed with a Student’s t test. n.s., not significant; *P < 0.05; **P < 0.01.