Figure 1.
STAT3 detection in the primary vaccinia lesion of infected SCID mice.
A) Slowly growing primary lesion in vaccinia infected SCID mice. B) STAT3 detected by immunohistochemistry in formalin fixed and paraffin embedded skin tissue from terminal vaccinia lesions of infected SCID mice (top panels) or uninfected SCID mouse (lower left). Lower right panel, normal rabbit serum negative control antibody detection in representative terminal vaccinia lesion. Scale bars, 100 µm.
Figure 2.
Topical application of Stattic accelerates vaccinia disease.
A) Schedule of SCID mouse experiments, in which Stattic or DMSO was applied topically for one week before and after ACAM-2000 scarification. B) Weight loss in ACAM-2000 infected SCID mice treated with STAT3 inhibitor stattic or vehicle control. Mean of 9–10 mice is shown. C) Survival proportions in ACAM-2000 infected SCID mice treated with STAT3 inhibitor stattic or vehicle control (n = 9–10 per group). Asterisk, p≤0.05. Representative of three experiments is shown.
Figure 3.
Topical STAT3 inhibition is associated with increased viral recovery.
STAT3 specific inhibitor stattic or DMSO was applied topically, for one week before and continuing through one week after scarification. A) Representative lesions in DMSO (upper row) and stattic (lower row) treated mice were photographed on day 13, and virus recovery on day 13 (C) was assessed in homogenized primary lesion tissue (n = 7–8). Asterisk, p≤0.01.
Figure 4.
Inhibitors of STAT3, caspase-1 and RIP1K enhance vaccinia virus infection and preserve the viability of keratinocytes, but not Vero cells.
A) Detection of luciferase activity over 24 hours post infection with ACAM-luc at 0.2 MOI in HEK001 and 308 keratinocytes, and Vero cells. Mean of replicate wells is plotted. B) Infection of keratinocytes (HEK001, 308) or Vero cells was performed with ACAM-2000 at 20 MOI. Viability at 6 hours post-infection was assessed by Alamar blue dye conversion (n = 4). C) Infection of HEK001 was performed with ACAM-2000 at 20 MOI, with vehicle control, Stattic, or caspase-3 inhibitor added. Viability at 6 and 12 hours post-infection was assessed by Alamar blue dye conversion (n = 4). D) Infection of keratinocytes (HEK001, 308) or Vero cells was performed with ACAM-2000 at 20 MOI, in the presence of DMSO vehicle control, STAT3 inhibitor, caspase-1 inhibitor, or Nec-1. Viability at 24 hours was assessed by Alamar blue dye conversion (n = 4). E) Keratinocytes (HEK001, 308) or Vero cells were infected with ACAM-luc at 0.2 MOI, in the presence of DMSO vehicle control, STAT3 inhibitor, Nec-1, or caspase-1 inhibitor. Luciferase activity at 24 hours is expressed as percent of uninfected control (n = 4). F) Differentiated primary keratinocytes at the air-liquid interface were infected with ACAM-luc at 20 MOI, in the presence of DMSO, STAT3 inhibitor, or Nec-1 (n = 2). Representative of triplicate experiments is shown. Asterisk, p≤0.05.
Figure 5.
Inhibitors of STAT3 and RIP1K, but not caspase-1, suppress inflammatory responses to vaccinia virus infection in keratinocytes.
HEK001 and 308 keratinocytes and Vero cells were transfected with reporter plasmids encoding luciferase under the control of promoters for NFκB (A), IFNβ (B), or ISRE (C). Cells were then challenged with UV inactivated ACAM-2000, or with infectious ACAM-2000 at 20 MOI combined with DMSO, Stattic, caspase-1 inhibitor, or Nec-1. Luciferase activity was detected at 24 hours post-infection (n = 4 wells per condition). Representative of triplicate experiments is shown. Asterisk, p≤0.05.
Figure 6.
Treatment of infected keratinocytes with STAT3-specific siRNA partially preserves cell viability and increases virus replication and inflammatory responses.
HEK001 cells were transfected with control or STAT3-specific siRNA preparations for 48 hours or treated with DMSO or Stattic immediately prior to infection. A) Wells were infected with ACAM-2000 at 20 MOI. Viability at 24 hours was assessed with Alamar blue fluorescence (n = 4). B) Wells were infected with ACAM-luc at 0.2 MOI. Cells were harvested at 24 hours, and luciferase in cell lysates was detected (n = 4). C) Cells were transfected with reporter plasmids encoding luciferase downstream of NFκB, IFNβ, or ISRE promoter elements. Transfected cells were infected with ACAM-2000 at 20 MOI, or with UV inactivated ACAM-2000. Luciferase signal was measured at 24 hours postinfection (n = 4). Representative of triplicate experiments is shown. D) HEK001 cells were transfected with scrambled control siRNA, or STAT3 directed siRNA. Cells were harvested at 48 hours and total mRNA and cDNA were prepared. Transcripts for -actin and STAT3 were quantified using qRT-PCR. Difference in threshold cycle number (Ct) between cells treated with control and STAT3 directed siRNA is shown (n = 9). E) HEK001 cells were mock transfected, or transfected with scrambled control siRNA or STAT3 directed siRNA. Cells were harvested at 48 hours. STAT3 and β-actin were detected in whole cell lysates by immunoblot. Relative intensity of STAT3 band, normalized to β-actin control band, was calculated using densitometric analysis for each treatment group. Representative of duplicate experiments is shown. Asterisk, p≤0.05.
Figure 7.
Treatment with Stattic reduces inflammatory responses in keratinocytes exposed to TLR ligands.
HEK001 cells were pretreated with Stattic or vehicle, then challenged with bacterial lipopolysaccharide (LPS), peptidoglycan (PGN) or S. typhimurium flagellin (FLA). TNF-α (A) and IL-β (B) in supernatants was assessed by ELISA at 24 and 48 hours. C) HEK001 cells treated with vehicle or Stattic were infected with ACAM-2000 at MOI = 20. Secreted TNF-α, IL-1β, and IL-6 were assessed at 12 hours (n = 2 wells per condition). Representative of triplicate experiments is shown. Asterisk, p≤0.05.
Figure 8.
Stattic reduces STAT3 and TAK1 detection in cytosol of infected keratinocytes.
Left: HEK001 keratinocytes were uninfected, or infected for 3 hours with ACAM-2000 at 20 MOI, in the presence of DMSO, Stattic, or Nec-1. At 3 hours cells were collected in cold hypotonic buffer. Left: Cytoplasmic fractions were evaluated for STAT3, TAK1, RIP1K, and housekeeping protein β-actin by immunoblot. Right: Relative intensity of STAT3 and TAK1 protein bands in infected, Stattic-treated wells, compared with infected, DMSO treated wells (n = 2 experiments). Asterisk, p≤0.05.