Role of chemokines in HPV-induced cancers.

Human papillomaviruses (HPVs) cause cancers of the uterine cervix, oropharynx, anus, and vulvovaginal tract. Low-risk HPVs, such as HPV6 and 11, can also cause benign mucosal lesions including genital warts, and in patients with recurrent respiratory papillomatosis, lesions in the larynx, and on occasion, in the lungs. However, both high and less tumorigenic HPVs share a striking commonality in manipulating both innate and adaptive immune responses in HPV- infected keratinocytes, the natural host for HPV infection. In addition, immune/inflammatory cell infiltration into the tumor microenvironment influences cancer growth and prognosis, and this process is tightly regulated by different chemokines. Chemokines are small proteins and exert their biological effects by binding with G protein-coupled chemokine receptors (GPCRs) that are found on the surfaces of select target cells. Chemokines are not only involved in the establishment of a pro-tumorigenic microenvironment and organ-directed metastases but also involved in disease progression through enhancing tumor cell growth and proliferation. Therefore, having a solid grasp on chemokines and immune checkpoint modulators can help in the treatment of these cancers. In this review, we discuss the recent advances on the expression patterns and regulation of the main chemokines found in HPV-induced cancers, and their effects on both immune and non-immune cells in these lesions. Importantly, we also present the current knowledge of therapeutic interventions on the expression of specific chemokine and their receptors that have been shown to influence the development and progression of HPV-induced cancers.

Toll-like receptor agonists, poly(I:C) and flagellin, lead to IL-36γ induction with divergent release kinetics and differentially alter autophagy in primary human keratinocytes

IL-36γ, a pro-inflammatory member of the IL-1 cytokine superfamily, can be induced and secreted by normal human foreskin keratinocytes (HFKs) in response to pathogenic stimuli, however, the mechanisms underlying the secretion are unknown. In this study, we demonstrate that stimulation with the TLR3 agonist, poly (I:C), led to a delayed secretion of IL-36γ compared to stimulation with the TLR5 agonist, flagellin, despite equal levels of the cytokine (p = 0.006). IL-36γ was shown to be released from HFKs in its inactive, uncleaved form, based on western blotting. Moreover, recombinant IL-36γ in its activated, cleaved form induced endogenous IL-36γ 10-fold (p = 0.004) and CXCL8 five-fold (p = 0.003) over baseline levels compared to unactivated full-length recombinant IL-36γ. The ratio of LC3b-II/LC3b-I was significantly higher in poly(I:C)-treated cells compared to flagellin-treated and unstimulated controls without a change in SQSTM1/p62 after 24 hours of stimulation (p = 0.043). Under fluorescence microscopy, poly(I:C) led to a two-fold increase at eight hours and four-fold increase at 24 hours in accumulated autophagosomes post-stimulation (p = 0.032). In contrast, autophagosomes were unchanged relative to baseline in response to flagellin. Bafilomycin A1 treatment enhanced poly(I:C)-mediated IL-36γ secretion (p = 0.044) while rapamycin led to a noticeable, but non-significant, increase in flagellin-mediated IL-36γ secretion, indicating that interrupting autophagic flux can alter IL-3γ grelease from HFKs. Finally, we show that, compared to clinically normal laryngeal tissue, there were significantly higher levels of LC3b-II in HPV-infected respiratory papilloma tissue, indicating a higher number of autophagosomes; a signature of disrupted autophagic flux.

Extracellular vesicles produced by primary human keratinocytes in response to TLR agonists induce stimulus-specific responses in antigen-presenting cells.

Cells can communicate through the extracellular vesicles (EVs) they secrete. Pathogen associated molecular patterns (PAMPs), alter the biophysical and communicative properties of EVs released from cells, but the functional consequences of these changes are unknown. Characterization of keratinocyte-derived EVs after poly(I:C) treatment (poly(I:C)-EVs) showed slight differences in levels of EV markers TSG101 and Alix, a loss of CD63 and were positive for autophagosome marker LC3b-II and the cytokine IL36γ compared to EVs from unstimulated keratinocytes (control-EVs). Flagellin treatment (flagellin-EVs) led to an EV marker profile like control-EVs but lacked LC3b-II. Flagellin-EVs also lacked IL-36γ despite nearly identical intracellular levels. While poly(I:C) treatment led to the clear emergence of a > 200 nm diameter EV sub-population, these were not found in flagellin-EVs. EV associated IL-36γ colocalized with LC3b-II in density gradient analysis, equilibrating to 1.10 g/mL, indicating a common EV species. Poly(I:C), but not flagellin, induced intracellular vesicles positive for IL-36γ, LC3b-II, Alix and TSG101, consistent with fusion of autophagosomes and multivesicular bodies. Simultaneous rapamycin and flagellin treatment induced similar intracellular vesicles but was insufficient for the release of IL-36γ+/LC3b-II+ EVs. Finally, a qRT-PCR array screen showed eight cytokine/chemokine transcripts were altered (p < 0.05) in monocyte-derived Langerhans cells (LCs) when stimulated with poly(I:C)-EVs while three were altered when LCs were stimulated with flagellin-EVs compared to control-EVs. After independent confirmation, poly(I:C)-EVs upregulated BMP6 (p = 0.035) and flagellin-EVs upregulated CXCL8 (p = 0.005), VEGFA (p = 0.018) and PTGS2 (p = 0.020) compared to control-EVs. We conclude that exogenous signals derived from pathogens can alter keratinocyte-mediated modulation of the local immune responses by inducing changes in the types of EVs secreted and responses in antigen presenting cells.

Poly(I:C) induces controlled release of IL-36γ from keratinocytes in the absence of cell death.

The epithelium is part of an integrated immune system where cytokines, toll-like receptors and their ligands, and extracellular vesicles play a crucial role in initiating an innate immune response. IL-36γ is a pro-inflammatory member of the IL-1 family that is mainly expressed by epithelial cells, but regulation of its expression and release are only beginning to be understood. Previous studies reported that IL-36γ is abundant in recurrent respiratory papillomatosis, a rare but devastating disease caused by human papillomaviruses (HPV) types 6 and 11, in which papillomas recurrently grow in and block the airway. Despite the overexpression of IL-36γ, papilloma tissues show no evidence of inflammation, possibly due to suppression of its release by HPVs. We have used primary human foreskin keratinocytes as a model to study IL-36γ regulation in normal epithelial cells. Low doses of poly(I:C) mediate expression and release of IL-36γ without inducing the cell death reported by those using high doses. PKR, an enzyme required for inflammasome activation, does not contribute to controlled release of IL36γ. The keratinocytes secrete IL-36γ in two forms, soluble and in extracellular vesicles. We conclude that there are two separately regulated pathways for the controlled secretion of IL-36γ from keratinocytes, which could contribute to the modulation of both local and systemic immune responses to viruses and other pathogens.