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. 1999 Apr;43(4):856-61.
doi: 10.1128/AAC.43.4.856.

The immune response modifier imiquimod requires STAT-1 for induction of interferon, interferon-stimulated genes, and interleukin-6

R L Bottrel  1 , Y L Yang, D E Levy, M Tomai, L F Reis
Affiliations

The immune response modifier imiquimod requires STAT-1 for induction of interferon, interferon-stimulated genes, and interleukin-6

R L Bottrel et al. Antimicrob Agents Chemother. 1999 Apr.

Abstract

Imiquimod is an oral inducer of interferon (IFN) and several other proinflammatory cytokines and has been successfully used topically as an antiviral agent for the treatment of genital warts. We have investigated the molecular mechanisms by which imiquimod induces the expression of IFNs, IFN-stimulated genes (ISGs), and proinflammatory cytokines in vivo, using mice deficient in various components of the IFN signaling system. Mice deficient in the transcription factor interferon regulatory factor 1 (IRF-1) or in the serine/threonine protein kinase PKR responded normally to imiquimod, producing high levels of circulating IFN and induction of several ISGs. On the other hand, when mice deficient in STAT-1 were treated, a 32-fold reduction in the level of circulating IFN was observed, together with a lack of induction of 2-5 oligo adenylate synthetase (2-5 OAS) and IRF-1 genes. Interestingly, there was also a lack of induction of interleukin-6 (IL-6) gene expression, although tumor necrosis factor was induced and readily detected in serum. In mice deficient in the type I IFN receptor, imiquimod induced levels of IFN similar to those in control mice, but again, neither 2-5 OAS, IRF-1, nor IL-6 genes were induced in mutant mice. Our results suggest that STAT-1 plays a critical role in the mechanism of gene activation by imiquimod. Moreover, induction of IL-6 gene expression appears to be dependent on components of the IFN signaling cascade.

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Figures

FIG. 1
FIG. 1
Levels of 2-5 OAS mRNA in organs of imiquimod-treated mice. Wild-type or IRF-10/0 mice were treated orally with imiquimod (1 mg/kg) for 4 h, and total RNAs from the organs indicated were collected and fractionated on a 1% agarose-formaldehyde gel. RNA was transferred to a nylon membrane and hybridized with a 2-5 OAS cDNA probe (31) labeled with [α-32P]dCTP by random priming (RediPrime; Amersham). After autoradiography, filters were stripped and hybridized with a 32P-labeled GAPDH cDNA probe (10) to control for RNA loading.
FIG. 2
FIG. 2
Levels of 2-5 OAS (A) and IRF-1 (B) mRNAs in the livers of imiquimod-treated mice. Wild-type or STAT-10/0 mice were treated orally with imiquimod (1 mg/kg) for 2 or 8 h, total RNAs were collected and fractionated on a 1% agarose-formaldehyde gel, and RNA was transferred to a nylon membrane. (A) The filter was hybridized with a 2-5 OAS cDNA probe (31). (B) The filter was hybridized with an IRF-1 cDNA probe (32) labeled with [α-32P]dCTP by random priming (RediPrime; Amersham). After autoradiography, filters were stripped and hybridized with a 32P-labeled GAPDH cDNA probe (10) to control for RNA loading.
FIG. 3
FIG. 3
Levels of 2-5 OAS and IRF-1 mRNAs in the spleens of imiquimod-treated mice. Wild-type or IFNR0/0 mice were treated orally with imiquimod (1 mg/kg) for 2 or 8 h. Total RNAs were collected from spleens and fractionated on a 1% agarose-formaldehyde gel, and RNAs were transferred to a nylon membrane. The filter was serially hybridized with [α-32P]dCTP-labeled cDNA probes for 2-5 OAS (31), IRF-1 (32), and GAPDH (10) (to control for equal RNA loading).
FIG. 4
FIG. 4
Levels of IL-6 mRNA in the spleens of imiquimod-treated mice. Wild-type, STAT-10/0 (A), or IFNR0/0 (B) mice were treated orally with imiquimod (1 mg/kg) for 2 or 8 h, total RNAs were collected and fractionated on a 1% agarose-formaldehyde gel, and RNAs were transferred to a nylon membrane. Filters were hybridized with an IL-6 cDNA probe (6) labeled with [α-32P]dCTP by random priming (RediPrime; Amersham). After autoradiography, filters were stripped and hybridized with a 32P-labeled GAPDH cDNA probe (10) to control for RNA loading.
FIG. 5
FIG. 5
Levels of circulating IL-6 (A), TNF (B), and IL-12 (C) in the sera of imiquimod-treated mice. Wild-type (WT) or STAT-10/0 (KO) mice were treated orally with imiquimod (1 mg/kg) for 2 or 8 h, and cytokine levels in the serum of each animal were measured by ELISA (R&D Systems).
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