Persistent human papillomaviruses (HPV) infecting basal keratinocytes (KCs) can lead to malignant transformation of epithelium through the accumulation of DNA mutations. Local immune suppression is often associated with HPV infection and epithelial transformation. We previously observed that Langerhans cells (LCs), the primary antigen-presenting cell of epithelium, are impaired in antigen presentation and the induction of adaptive immunity in the transgenic K14E7 mouse model of HPV-induced dysplastic epithelium. LC impairment is correlated with reduced production of interleukin 34 (IL-34), a cytokine expressed by KCs and a critical molecule for LC homeostasis. Low expression of IL-34 is also associated with poor prognosis in epithelial cancers. The objectives of this study were to decipher the transcriptional regulation of IL-34 in hyperproliferative epithelial cells and develop tools to restore its expression in hyperplastic epithelium.
Using single-cell RNA sequencing to investigate the expression of IL-34 in hyperplastic K14E7 skin, we found a significant reduction of IL-34 in KCs, which was confirmed by PCR and ELISA. Employing the Assay for Transposase-Accessible Chromatin sequencing (ATAC-seq), we investigate the changes in transcriptional regulation of KCs and LCs in K14E7 epithelium. Our analysis revealed more than 30,000 significant differentially enriched sites (DES) in each cell type, with 9 DES in chromatin accessibility identified at the Il34 locus. Of these, 2 DES were in proximity to the skin-specific promoter of Il34. This suggests that altered transcriptional regulation of Il34 likely underpins the observed reduction in transcript and protein content and implying a difference in occupancy of site-specific transcription factors. In addition, we also identified 6 DES at the Csf1r locus. To test whether the restoration of IL-34 in hyperproliferative epithelium can repair the function of LCs and subsequent adaptive immune responses, we constructed lentiviruses encoding Il34 (LV-Il34). Delivering LV-Il34 intradermally into the K14E7 and WT ear pinnae, we found that 1.5 x 10^6 TU of the generated LV-Il34 was able to restore IL-34 levels in K14E7 ear skin back to normal physiological levels. LV-Il34 is a promising tool for further investigations into the role of IL-34 in skin immunity and its potential use as gene therapy for epithelial cancers.