Frontiers in immunology · 2026
Comparative analysis of the impact of interferon regulatory factors on basal and induced interferon and interferon stimulated gene expression in human and canine keratinocytes.
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Quinlan Sarah, May Susan, Weeks Ryan, Luff Jennifer
Аннотация
INTRODUCTION: Keratinocytes are key barrier cells able to mount a robust interferon (IFN) antiviral response to defend against infection in the skin. Similar to humans, dogs spontaneously develop skin disorders associated with dysregulation of IFN immunity and can be used as a large animal model to investigate these diseases. One of the critical factors driving IFN regulation are interferon regulatory factors (IRFs). IRFs are crucial in upregulating the antiviral type I or type III IFNs, which then subsequently upregulate hundreds of antiviral effector proteins called interferon stimulated genes (ISGs).
METHODS: We sought to comparatively analyze how IRF1, 3 and 7 regulate type I and type III IFNs and ISGs using canine and human keratinocyte cultures. To this aim, we stimulated keratinocytes in vitro with dsDNA and dsRNA ligands and analyzed upregulation of IFNs and ISGs using real time quantitative PCR (RT-qPCR). Knockout of IRF3 and IRF1 and knockdown of IRF7 was performed on dog and human keratinocytes using CRISPR-Cas9 and shRNA methods, respectively. Knockdown or knockout was confirmed using RT-qPCR and/or western blots.
RESULTS: We demonstrated that compared to canine keratinocytes, human keratinocytes express higher basal type I IFN-β and ISGs, induce higher levels of type III IFNs upon stimulation, and overall, express higher IFN and ISG copies. We further showed that IRF regulation of induced IFNs, particularly IRF1, can be species-specific and/or stimulus-specific, and putatively that non-IRF mechanisms likely mediate basal expression of type I IFNs in human keratinocytes and type III IFNs in canine keratinocytes. While both IRF3 and IRF7 are critical for type I and III IFN induction following activation of cytosolic dsDNA and dsRNA receptors, neither can account for differences in the induced type I versus III IFN expression between dog and human, suggesting non-IRF1, 3, or 7 mechanisms underly this regulation.
CONCLUSIONS: These studies provide support for use of the dog as a model to discern mechanisms underlying high basal type I and ISGs in human keratinocytes, how high basal ISGs can modulate the antiviral response, and uncover non-IRF mediated mechanisms regulating the type I versus type III response in keratinocytes.
METHODS: We sought to comparatively analyze how IRF1, 3 and 7 regulate type I and type III IFNs and ISGs using canine and human keratinocyte cultures. To this aim, we stimulated keratinocytes in vitro with dsDNA and dsRNA ligands and analyzed upregulation of IFNs and ISGs using real time quantitative PCR (RT-qPCR). Knockout of IRF3 and IRF1 and knockdown of IRF7 was performed on dog and human keratinocytes using CRISPR-Cas9 and shRNA methods, respectively. Knockdown or knockout was confirmed using RT-qPCR and/or western blots.
RESULTS: We demonstrated that compared to canine keratinocytes, human keratinocytes express higher basal type I IFN-β and ISGs, induce higher levels of type III IFNs upon stimulation, and overall, express higher IFN and ISG copies. We further showed that IRF regulation of induced IFNs, particularly IRF1, can be species-specific and/or stimulus-specific, and putatively that non-IRF mechanisms likely mediate basal expression of type I IFNs in human keratinocytes and type III IFNs in canine keratinocytes. While both IRF3 and IRF7 are critical for type I and III IFN induction following activation of cytosolic dsDNA and dsRNA receptors, neither can account for differences in the induced type I versus III IFN expression between dog and human, suggesting non-IRF1, 3, or 7 mechanisms underly this regulation.
CONCLUSIONS: These studies provide support for use of the dog as a model to discern mechanisms underlying high basal type I and ISGs in human keratinocytes, how high basal ISGs can modulate the antiviral response, and uncover non-IRF mediated mechanisms regulating the type I versus type III response in keratinocytes.