Altered Toll-like receptor expression and function in HPV-associated oropharyngeal carcinoma.

Priscila Lie Tobouti,Keith D Hunter,Raghu Radhakrishnan,Suzana Cantanhede Orsini Machado De Sousa,Robert Bolt

doi:10.18632/oncotarget.18959

Abstract

Toll-like receptors (TLRs) have been widely investigated due to their importance in the inflammatory response and possible links to tumor promotion/regression and prognosis. In cancers with an infective etiology, such as human papillomavirus (HPV)-associated Oropharyngeal Squamous Cell Carcinoma (OPSCC), TLR responses may be activated and play a role in tumorigenesis. Our aim was to assess the expression of all TLRs in OPSCC cell lines (both HPV+ and HPV–) by qPCR, Western Blot and flow cytometry and assess their response to TLR ligands lipopolysaccharide (LPS), LPS ultra-pure (LPS-UP) and peptidoglycan (PGN) by analyzing IL-8 and IL-6 production. We also immunostained 61 OPSCC tissue samples with anti-TLR4. Results showed lower TLR1 and TLR6 mRNA expression and higher TLR9 protein expression in HPV+ when compared to HPV–OPSCC cells. TLR4 expression did not vary by HPV status in OPSCC cells, but TLR4 expression was significantly lower in HPV+OPSCC tissues. After stimulation with PGN, only one cell line (HPV+) did not secrete IL-6 or IL-8. Furthermore, HPV+OPSCC lines showed no IL-6 or IL-8 production on treatment with LPS/LPS-UP. The data suggest changes in TLR4 signaling in HPV+OPSCC, since we have shown lower tissue expression of TLR4 and no pro-inflammatory response after stimulation with LPS and LPS-UP. Also, it suggests that OPSCC may respond to HPV infection by increased expression of TLR9. This study demonstrates differences in expression and function of TLRs in OPSCC, which are dependent on HPV status, and may indicate subversion of the innate immune response by HPV infection.

Highlights

The innate immune response detects pathogenic microorganisms through a number of mechanisms, including recognition of pathogen-associated molecular patterns (PAMP) by pattern-recognition receptors (PRRs), which include the Toll-like receptors (TLR) [1]
Despite differences in mRNA expression, there were no significant differences in TLR1 or TLR6 protein expression when comparing human papillomavirus (HPV)+ and HPV– cell lines, as measured by flow cytometry (Figure 1B)
In this study, where we evaluated TLR 1-10 mRNA expression by qPCR, we observed that only TLR1 and TLR6 mRNA are more expressed in HPV– when compared to HPV+ cell lines

Summary

Introduction

The innate immune response detects pathogenic microorganisms through a number of mechanisms, including recognition of pathogen-associated molecular patterns (PAMP) by pattern-recognition receptors (PRRs), which include the Toll-like receptors (TLR) [1]. The near-ubiquitous nature of TLR expression within normal epithelia relates to its important barrier function against invading microorganisms; TLR activity is essential for an effective host response to be mounted [3]. In addition to this central role in protection against infection, TLRs have roles in maintaining tissue homeostasis through the regulation of inflammatory and reparative responses to tissue injury [4]. The link between the immune system and cancer progression has led several groups to assess the role of receptors capable of activating signaling pathways for the recruitment of inflammatory cells, among which are included the TLRs

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