Treatment-recalcitrant chronic rhinosinusitis with polyps is associated with altered epithelial cell expression of interleukin-33

Abstract

Background: Abnormalities in host mucosal immunity exist in chronic rhinosinusitis with nasal polyps (CRSwNPs), but it is unclear whether this is a cause or an effect of the eosinophilic inflammation and frequent microbial colonization that characterizes the disease. Sinonasal epithelial cells (SNECs) are critical participants in healthy antimicrobial innate immune defense. They also can promote Th2 inflammation with various mediators, including interleukin (IL)-33, which induces T helper cells to produce Th2 cytokines.

Methods: CRSwNP SNECs were obtained during sinus surgery and stored. Patients were subsequently classified as either treatment responsive or treatment recalcitrant, based on long-term outcomes of medical and surgical therapy. Epithelial cells from these patients were grown in air-liquid interface (ALI) culture and treated with IL-13, as well as the bacteria-associated molecule, CpG. Expression of IL-33 mRNA was determined by real-time polymerase chain reaction.

Results: Recalcitrant CRSwNP epithelial cells had increased baseline expression of IL-33 compared with responsive CRSwNPs, which was further increased by 24-hour exposure to CpG. Treatment-responsive epithelial cells were not induced by CpG to express IL-33. Prolonged treatment with IL-13 during differentiation at the ALI diminished the baseline expression of IL-33 and prevented the subsequent induction of IL-33 by CpG.

Conclusion: Mucosal innate immunity likely plays an important role in CRSwNP pathogenesis. A definitive link between infectious triggers and the development of Th2 inflammation has been elusive. We have found constitutive IL-33 expression by SNECs in recalcitrant CRSwNPs, which can be further induced by a bacteria-associated molecular pattern. Dysregulated epithelial cell immune interactions between host and environment may contribute to Th2 inflammation in CRSwNPs.

Figure 1

Expression of interleukin (IL)-33 mRNA was assessed by real-time polymerase chain reaction (PCR) in primary cultures of sinonasal epithelial cells (SNECs) derived from chronic rhinosinusitis with nasal polyps (CRSwNP) patients (n = 32). The baseline level of IL-33 mRNA was threefold greater in epithelial cells from patients with recalcitrant CRSwNP (n = 11) who continue to have nasal polyps despite sinus surgery and continuous medical therapy. Error bars denote standard error of the mean (*p = 0.001). Increased expression of IL-33 mRNA in cultured SNECs obtained from patients with treatment-recalcitrant CRSwNPs.

Figure 2

In differentiated sinonasal epithelial cells (SNECs), exposure to the bacterial pathogen-associated molecule CpG (10 μM) for 24 hours induces increased expression of interleukin (IL)-33 mRNA only in cells derived from patients with recalcitrant chronic rhinosinusitis with nasal polyps (CRswNPs). Each symbol denotes an individual subject’s SNECs (n = 5 for each group). There was a threefold induction for the recalcitrant group as a whole. CpG induces increased expression of IL-33 mRNA in differentiated SNECs derived from patients with recalcitrant CRswNP.

Figure 3

Effect of 10-day exposure to interleukin (IL)-13 on differentiating sinonasal epithelial cells (SNECs). (A) Confocal microscopy of differentiated epithelial cell cultures after 21 days at the air–liquid interface (ALI). The green label shows β-IV-tubulin expression denoting cilia, and the red label shows the cell boundaries with the marker Zo-1, which labels intercellular tight junctions. The top left panel shows the distribution of ciliated cells without IL-13 treatment and the bottom panel shows the effect of IL-13 exposure during days 3–13 of differentiation. (B) Real-time polymerase chain reaction (PCR) shows decreased β-IV-tubulin mRNA expression in cells exposed to IL-13 for 10 days during differentiation. (C) Real-time PCR reveals a 3.65-fold decreased expression of IL-33 by cells exposed to IL-13 during differentiation when compared with unexposed cells derived from the same patients. Error bars denote standard error of the mean (*p = 0.03). Effect of IL-13 exposure during epithelial cell differentiation at the ALI on ciliation (panels A and B) and level of IL-33 mRNA expression (panel C).