CRS is a general term for what appears to be numerous distinct pathways to inflammation of the paranasal sinuses for which the distinction between the presence or lack of polyps can be of significant therapeutic benefit.22
However, even within CRSwNP, multiple subsets of disease with respectively different treatments exist including allergic fungal sinusitis and aspirin-exacerbated respiratory disease. Nevertheless, polyps characterized by eosinophilia are only seen ~80% of the time in western cultures23
and perhaps substantially less in certain Asian populations.24,25
The remaining portion of patients has NPs that are significantly lacking in an eosinophilic infiltration, but this entity has not been subject to the same level of scrutiny as the eosinophilic variety as we sought to provide with the current study and microarray analysis.
Several studies using microarray technology in the evaluation of NPs have been published in recent years. Benson and colleagues have published several articles regarding their initial study on the effects of corticosteroids on the gene profile of NPs.14
Later, Fritz and colleagues15
and Figuerido et al.12
compared polyp tissue with adjacent inflamed mucosa and showed significant differences in the gene expression profiles. Liu et al.26
and Wang et al.16
also found differences comparing polyps with sphenoid sinus mucosa and inferior turbinate mucosa, respectively. However, these several studies all evaluated CRSwNP in general, without specific attention to any subset therein. Orlandi et al.
on the other hand, did evaluate subsets of CRSwNP, but unlike our study, theirs focused on two variants of eosinophilic CRSwNP (E-CRSwNP). As such, no study has yet specifically explored NE-CRSwNP through either microarray technology or extensive RT-PCR of genetic transcripts.
In this study, we found 120 genes that were either significantly differentially expressed in polyp tissue when compared with normal sinus tissue controls, but the various inflammatory cytokines typically seen in CRS were not found to have a significant change in regulation. This may reflect the inherent variability of gene expression and the heterogeneity of this disorder in addition to the low power analysis of the small sample size used for this screening technique. These problems seem to be common to microarray analysis and other authors have indicated that this can lead to both spurious data as well as underrecognizing important differences.27
Pooling of specimens may have been able to compensate for this problem, but this method has also been discouraged elsewhere.28
Some trends were, however, observed with these cytokines and several of these were selected for further study with RT-PCR. Although not every prediction was confirmed, the data were of importance. Most compelling was the lack of evidence for expression of genes associated with allergic inflammation including Th2-associated cytokines (e.g.
, IL-4 and IL-13), consistent with our belief that this is a unique disorder and the down-regulation of genes associated with eosinophilic inflammation (eotaxin and eosinophil cationic protein) on the gene array. In contrast, we did see a fourfold elevation of SCF (KITLG). As a stimulator of the differentiation and proliferation of mast cells, its up-regulation in the polyp tissue is consistent with our previous histological findings of the presence of mast cells in this tissue. The fibrosis in these polyps may well be mediated through this type of mast cell process, which has been implicated in other fibrotic diseases such as rheumatoid arthritis, scleroderma, and idiopathic pulmonary fibrosis.28
Both transcripts and protein levels for IL-8 (CXCL8) and CXCL1 were also noted to be significantly elevated in the polyp tissue. These chemokines are both attractants for neutrophils indicating a role for this immune cell in the disease. Although neutrophils were not seen in our histological samples, it may be that polyp formation in this disease develops in the setting of chronic infection, biofilm formation, and purulent exudates that are remote from the polyp.
As discussed, our previous studies indicated an increased fibrotic stroma in these types of polyps and the nearly 10-fold increase in tenascin-C in the qRT-PCR is consistent with this observation. Tenascin-C is often transiently expressed in acute tissue injury and inflammation where it regulates fibroblast migration.29
Its presence in a chronic disease such as NECRSwNP implies a “persistent acute” injury to the tissue as a part of the overall disease process resulting in a more fibrotic appearing polyp.
The role that hypoxia might play in upper airway inflammatory disease is a potentially important one.30
HIF-1α is an inducible transcription factor expressed in hypoxic conditions that is involved in activation of glycolytic and inflammatory pathways.31
NP-derived fibroblasts have been specifically shown to increase their production of HIF-1α in response to hypoxic conditions32
and a corresponding in vivo
up-regulation was significantly shown in this study, consistent with the reported hypoxic milieu observed in CRS.33
Although the increased expression of HIF-1α may simply be a stress response, the implication can not be ignored that hypoxia may play a significant role in the pathogenesis of NE-CRSwNP. Improved aeration with a secondary reduction of HIF-1α could contribute to the therapeutic benefit observed after surgery.
Thymus and activation-regulated chemokine and thymic stromal lymphoprotein were not found to be significantly up-regulated and IL-4, IL-13, and IFN-γ, the hallmark cytokines for Th2- and Th1-mediated disease, respectively, were found to be down-regulated. This correlates well with a recent study by Kim et al.23
where on immunohistochemical study of NE polyps, 0/20 expressed only CCR5+
(Th1) cells, 6/20 expressed only CCR3+
(Th2) cells, 4/20 expressed both, and 10/20 (50%) expressed neither Th1 or Th2 cells.
Our investigation also found the expression of two other genes to be substantially increased on both microarray and RT-PCR data, ABCB11 and AMFR. The former is a bile salt exporter linked to drug resistance and for which studies investigating its presence in hepatic inflammation and stimulation with IL-6 have actually shown a down-regulation.34
It is not immediately clear why this gene was found to be up-regulated in sinus tissues, especially under a condition of IL-6 up-regulation, and as such, this deserves additional evaluation in future studies.
AMFR, on the other hand, is a gene that codes for the receptor for autocrine motility factor, a cytokine seen to be produced by tumor cells,35
and its up-regulation in our study correlates well with other studies on NPs indicating up-regulation of carcinogenic genes.14,15
Although a large differential expression of SPINK7 and CLCA2 was seen during microarray analysis, this was not confirmed by RT-PCR.
When viewed in the context of the RT-PCR results, the predicted direction of change for the studied genes was confirmed in 11/16 (68.8%). It is uncertain as to why a greater correlation was not seen. Perhaps this relates to the inability for population estimates to be made based on a very limited sample size, especially in what may be a heterogeneous entity. Nevertheless, the microarray was able to provide some very useful confirmable data regarding some of the novel genes identified.