Decreased Langerhans cell responses to IL-36γ: altered innate immunity in patients with recurrent respiratory papillomatosis

Abstract

Recurrent respiratory papillomatosis (RRP) is a rare, chronic disease caused by human papillomaviruses (HPVs) types 6 and 11 that is characterized by the polarization of adaptive immune responses that support persistent HPV infection. Respiratory papillomas express elevated mRNA levels of IL-36γ, a proinflammatory cytokine in comparison to autologous clinically normal laryngeal tissues; however there is no evidence of inflammation in these lesions. Consistent with this, respiratory papillomas do not contain TH1-like CD4(+) T-cells or cytotoxic CD8(+) T-cells, but instead contain a predominance of TH2-like and T regulatory cells (Tregs). In addition, papillomas also are infiltrated with immature Langerhans cells (iLCs). In this study, we show that papilloma cells express IL-36γ protein, and that human keratinocytes transduced with HPV11 have reduced IL-36γ secretion. We now provide the first evidence that peripheral blood-derived iLCs respond to IL-36γ by expressing inflammatory cytokines and chemokines. When stimulated with IL-36γ, iLCs from patients with RRP had lower expression levels of the TH2-like chemokine CCL-20 as compared with controls. Patients' iLCs also had decreased steady state levels of CCL-1, which is a proinflammatory chemokine. Moreover, CCL-1 levels in iLCs inversely correlated with the severity of RRP. The combined decrease of TH1- and a TH2-like chemokines by iLCs from patients could have consequences in the priming of IFN-γ expression by CD8(+) T-cells. Taken together, our results suggest that, in RRP, there is a defect in the proinflammatory innate immune responses made by iLCs in response to IL-36γ. The consequence of this defect may lead to persistent HPV infection by failing to support an effective HPV-specific, TH1-like and/or Tc1-like adaptive response, thus resulting in the predominant TH2-like and/or Treg micromilieu present in papillomas.

Figure 1

IL-36γ protein is overexpressed in papilloma tissues. Western blot showing IL-36γ expression levels in papilloma tissue (P) and autologous clinically normal laryngeal tissue (CN) from RRP patients. β-actin (lower panel) shown as a loading control. Representative of six matched pairs of tissues.

Figure 2

HPV-11 suppresses release of IL-36γ from keratinocytes. Human foreskin keratinocytes that had been transduced with either a lentivirus construct expressing all early HPV-11 genes or the empty vector were treated with poly(I:C) or solvent for 96 h. IL-36γ release was measured by ELISA (A), with results shown as the mean ± SD of three assays, *p < 0.05. Comparable levels of intracellular IL-36γ were confirmed by Western blot (B).

Figure 3

Papillomas contain abundant iLCs. iLCs were CD1a⁺ (A,B) and langerin⁺ (C,D), but CD83⁻ (E). A positive control (lymphoma) was CD83⁺ (F). (A–B) Paraffin sections, DAB stain. (C–D) frozen sections, AEC stain. (E–F) Paraffin sections, immunofluorescence with DAPI counterstain. (G, H) Flow cytometry of LCs isolated from papilloma biopsies. LCs (G, lower right) were approximately half as frequent as T-cells (G, upper left). LCs were CD14 negative (H, lower right), confirming that they were not monocytes and that there was no significant contamination by blood.

Figure 4

Characterization of LCs generated from peripheral blood monocytes. iLCs were generated from isolated peripheral blood monocytes by incubating blood monocytes with GMCSF, TGF-β1, and IL-4. After 6 d, they were analyzed by flow cytometry. The cells were E-cadherin⁺ and approximately 50% were langerin⁺ (A), but immature (CD83⁻) (B). Day 11 iLCs (C) could be induced to mature, as measured by surface expression of CD83 and HLADR, when stimulated with either IL-36γ (D) or LPS (E) for 48 h.

Figure 5

Response of iLCs to IL-36γ. Cells were stimulated with either recombinant inactive, full-length (aa 1–169) IL-36γ (control) or the active, shorter form (aa 18–169) of IL-36γ for 4 h. Induction of mRNA for specific proinflammatory cytokines (A) and chemokines (B) was measured by q-PCR. Results are the mean ± SD of at least six separate cultures, normalized to control cultures treated with solvent.

Figure 6

Differentiation of LCs from monocytes does not alter IL-1 receptor and IL-36 receptor expression. Blood-derived monocytes (d 2) and iLCs derived from monoctyes (d 6) were assayed for mRNA expression of IL-1R1, IL-1R3, and IL-1R6 and compared with GAPDH mRNA expression (δCT).

Figure 7

iLCs from patients are less responsive to IL-36γ. Cells from both healthy controls and RRP patients were stimulated with active IL-36γ for 4 h, and increased expression of proinflammatory cytokines and chemokines measured by q-PCR. A consistent pattern of blunted expression by the patient-derived LCs was evident, with the difference in CCL20 significant and the difference in IL-1β just missing significance. Results are the mean ± SD of at least six separate iLC cultures from controls and seven isolates from patients. *p < 0.05.

Figure 8

Expression of CCL1 in iLCs correlates with severity of RRP. The level of constitutive CCL1 expression in iLCs from patients with RRP was determined by q-PCR. Expression levels in iLCs from eight controls were defined as 100%; and individual samples clustered tightly around the 100% value (not shown); there was nearly a two-log reduction in expression as severity increased in the patients, p < 0.0001. Disease severity score is a composite value of the rate of regrowth of papilloma mass divided by the time in days since the previous surgery. A score over 0.06 is classified as severe disease.