BACKGROUND: Chronic rhinosinusitis (CRS) is a nasal and sinus mucosa chronic inflammation of at least 12 weeks duration with occasional exacerbations. CRS can be further subdivided into chronic rhinosinusitis with nasal polyps (CRSwNP) and chronic rhinosinusitis without nasal polyps (CRSsNP). Furthermore, at least 20 % of CRS patients suffer from uncontrolled CRS with persistent bothersome symptoms ( ⡥5 score on the visual analog scale, VAS 0-10) and recurrences after endoscopic surgeries despite appropriate treatment. The CRS mucosal immunophenotypes – endotypes diversity is still not well characterized. Importantly, the uncontrolled CRS - associated mucosal T cells are unknown.
OBJECTIVES: We aimed to evaluate the different mucosal T cell subtypes in yet untreated, steroid naïve patients with CRSwNP and patients with CRSsNP. In contrast to previous studies, we sought to identify the characteristic nasal mucosa T cells in patients with uncontrolled CRSwNP and CRSsNP and in patients with well-controlled CRSsNP and CRSsNP. We focused on cytotoxic CD8+ and double-negative (DN) CD4-CD8- T cells, which have not been described in CRS yet. Additionally, we aimed to evaluate the master transcription factors gene expression levels of T cell subtypes in CRSwNP and CRSsNP that could represent new, up-stream targets for DNAzyme topical nasal mucosa treatment to suppress the inflammation.
MATERIALS AND METHODS: 31 untreated, steroid-naïve CRS patients (18 CRSwNP, 13 CRSsNP) with severe symptoms were prospectively included. Biopsy was taken from the middle meatal nasal polyp in CRSwNP patients and the uncinate process mucosa in CRSsNP patients. The biopsies were examined histopathologically and analyzed by flow cytometry for T cell subtypes (Th1, Th2, Th17, Tc1, Tc17 and DN T cells), activation markers (CD69, CD25, HLA-DR) and cytotoxicity markers (CD28, CD27). Additionally, in 22 CRS patients (14 CRSwNP and 8 CRSsNP), the gene expression levels of major transcription factors T-box transcription factor (T-bet, TBX21), GATA binding protein 3 (GATA3), Retinoic acid-related orphan receptor C (RORC) and Forkhead box P3 (FOXP3) were measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Patients were followed up on average from 24 to 36 months with repeated visits. 5 of 18 CRSwNP and 5 of 13 CRSsNP had uncontrolled CRS despite extensive medical and surgical treatment. The data generated in the study were analyzed using GraphPad Prism 6.0.
RESULTS: Significantly more CRSwNP patients compared to CRSsNP patients had tissue eosinophilia >10 per HPF. CRSwNP mucosa was characterized by higher CD4+ cell numbers and more abundant activated CD4+CD25+ cells, suggesting Th-driven inflammation. We also observed increased Th1 cells in CRSwNP; however, we did not find any impact of those cells on the CRSwNP disease control. CRSsNP mucosa was characterized by higher CD8+ T cells and abundant effector cytotoxic CD8+CD28-CD27- cells with simultaneously decreased central memory CD8+CD28+CD27+ cell numbers, speaking for cytotoxic inflammation type in patients with CRSsNP. Furthermore, this was confirmed with even more abundant mucosal CD8+ cells in patients with uncontrolled CRSsNP compared to patients with well-controlled CRSsNP. Importantly, in both CRSwNP and CRSsNP, we found surprisingly high percentages (10-20%) of mucosal DN T cells in contrast to normal 1-5% range. Additionally, higher numbers of effector cytotoxic CD28-CD27- DN T cells and a lower number of central memory CD28+CD27+ DN T cells were observed. According to our knowledge, this is the first study to demonstrate such high numbers of DN T cells in CRS mucosa. We demonstrated 3-fold higher numbers of proinflammatory DN T cells and lower count of partly differentiated, effector memory CD28-CD27+ cells in patients with uncontrolled CRSwNP compared to patients with well-controlled CRSwNP. The source of DN T cells in CRS is currently unknown; however, DN T cells might also be an important producer of IL-5, the major type 2 cytokine. Well-controlled CRSwNP was characterized by a higher number of Th17 cells that probably represent a normal homeostatic immune response in healthy nasal mucosa. In patients with well-controlled CRSsNP, we demonstrated higher numbers of DN T cells, which could be suppressive regulatory and could be able to limit harmful CD8+ T cell response and mucosal damage in these patients. In patients with eosinophilic CRSwNP, we confirmed the type 2 inflammation by the higher level of GATA3 gene expression compared to patients with noneosinophilic CRSwNP. However, activated effector DN T cells can also secrete type 2 cytokines, although their master transcription factor is currently unknown. In patients with CRSsNP, we unexpectedly found simultaneous upregulation of T-bet, GATA3 and RORC gene expression levels in comparison to patients with CRSwNP; we might speculate here, that these high gene expression levels of all three effector T cells’ master transcription factors originate from expanded effector cytotoxic, activated CD8+ cells in CRSsNP mucosa.
CONCLUSIONS: We demonstrated distinct nasal mucosa T cell patterns for each CRS type: elevated CD4+ T cells in patients with CRSwNP and abundant CD8+ T cells with higher effector cytotoxic CD8+CD28-CD27- type in patients with CRSsNP. Increased mucosal DN T cells were found for the first time in patients with CRS. Moreover, those DN T cells were of elevated effector cytotoxic type. The present study was the first to analyze T cell subtypes in association with CRS disease control. The 3-times higher count of potent effector activated DN T cells in patients with uncontrolled CRSwNP might play a central role in uncontrolled CRSwNP pathogenesis, could represent a new
treatment target and therefore, should be further investigated. In contrast, DN T cells might be of the regulatory suppressive type in patients with CRSsNP, limiting the harmful cytotoxic mucosal inflammation. The unexpectedly found simultaneous upregulation of all three effector T cells’ master transcription factors in CRSsNP patients was a sign of expanded activated effector T cells in CRSsNP mucosa and is currently unexplained; however, it might originate from abundant effector cytotoxic CD8+ cells. Our results are scientifically as well as clinically applicable. In the perspective of future transcription factor-targeted topical nasal treatment development, the simultaneous coexpression of two different major transcription factors in effector or regulatory T cells should be taken into account. The understanding of inflammation pathogenesis in the nasal mucosa of patients with CRSwNP and patients with CRSsNP will upgrade the future CRS diagnostics with biomarkers, modify our current treatment algorithms and make the personalized medicine for patients with CRS possible. In the future, specific CRS mucosal immunophenotypes – endotypes will apply for the choice of the medications and tailor the extent of endoscopic sinus surgery that would gradually not follow the same standards for all anymore. Patient-tailored treatment will especially be important for uncontrolled CRS patients.
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