Altered expression of genes associated with innate immunity and inflammation in recalcitrant rhinosinusitis with polyps

Abstract

Background: The role of the innate immune system in the pathophysiology of chronic rhinosinusitis (CRS) is poorly understood. In this study, we compared sinonasal expression of toll-like receptors (TLRs), complement components, serum amyloid A, and inflammatory genes (chemokines and cytokines) in control subjects and patients undergoing sinus surgery for CRS.

Methods: Eleven control subjects and 30 subjects with CRS unresponsive to medical management were enrolled prospectively before undergoing endoscopic sinus surgery. Ethmoid mucosal specimens were obtained surgically and processed for RNA extraction. Real-time polymerase chain reaction was used to quantitate the level of expression of messenger RNA (mRNA) for TLR, acute phase proteins, and cytokine genes. Subjects were followed for a minimum of 6 months postoperatively with nasal endoscopy to assess for recurrence of polyps.

Results: mRNA for all target genes was detected in the ethmoid mucosa of both control and CRS subjects. The level of gene expression was normalized to the housekeeping genes 18s RNA and glyceraldehyde-3-phosphate dehydrogenase. As compared with controls, CRS was associated with significantly higher expression of TLR2 and the inflammatory genes macrophage-inflammatory protein alpha, RANTES, and granulocyte-macrophage colony-stimulating factor. Patients with early recurrence of polyps after surgery had significantly decreased expression of TLR2, 9, and serum amyloid A and increased expression of macrophage-inflammatory protein alpha compared with surgery-responsive patients.

Conclusion: This study shows the increased levels of expression of TLR2 and a variety of inflammatory genes in sinonasal mucosa of CRS patients compared with controls. Whether these differences play a role in pathogenesis or are merely manifestations of disease activity is worthy of investigation.

Figure 1

Standard procedure for obtaining uncinate mucosa endoscopic sinus surgery. The uncinate process was resected in an atraumatic fashion and immediately preserved in RNAlater solution. UNC, uncinate process; MT, middle turbinate; BE, bulla ethmoidalis.

Figure 2

Representative examples of responsive and recalcitrant CRS. (A) Responsive CRSwNP prior to surgical management. Left nasal cavity is shown (*nasal polyps). (B) Same sinonasal cavity 6 months postsurgery (MAX, maxillary sinus; SPH, sphenoid sinus). (C) Recalcitrant CRSwNP 9 months postsurgery. The right nasal cavity is shown (MT, middle turbinate).

Figure 3

RT-PCR comparison of (A) housekeeping genes and TLR or (B) host defense and inflammatory genes in control (light bars) and CRS patients (dark bars). Values are C_T; a lower value is a higher level of expression and each unit represents a twofold change. Figure shows increased expression of TLR2 and GM-CSF in CRS patients (*p < 0.05; TLR2, p < 0.04; GM-CSF, p = 0.04).

Figure 4

RT-PCR comparison (A) of the expression of TLR and (B) acute phase proteins and inflammatory genes in control (light bars) and CRS patients (dark bars). Values are ΔC_T, normalized to the housekeeping gene 18s RNA; a lower ΔC_T value corresponds to a higher level of expression. Figure shows increased expression of TLR2, MIP-1α, RANTES, and GM-CSF in sinonasal tissue from CRS patients (*p < 0.05; TLR2, p = 0.05; MIP-1α, p = 0.01; RANTES, p = 0.03; GM-CSF, p = 0.02).

Figure 5

(A) RT-PCR comparison of the expression of TLR and acute phase protein genes in treatment-responsive (light bars) and recalcitrant CRS (dark bars) samples. Values are ΔC_T, normalized to the housekeeping gene GAPDH; a lower ΔC_T value corresponds to a higher level of expression. Figure shows decreased expression of TLR2, TLR9, and SAA in sinonasal tissue from CRS patients (*p < 0.05; TLR2, p = 0.003; TLR9, p = 0.002; SAA, p = 0.04). (B) RT-PCR comparison of expression of inflammatory genes in treatment-responsive (light bars) and recalcitrant CRS (dark bars) samples. Values are ΔC_T, normalized to the housekeeping gene GAPDH; a lower ΔC_T value corresponds to a higher level of expression. Figure shows increased expression of MIP-1α in sinonasal tissue from CRS patients with recalcitrant disease (*p < 0.05, MIP-1α, p < 0.05).