Type 2 inflammation suppression by T regulatory cells attenuates the eosinophil recruitment in mucosa of chronic sinusitis.

Type 2 inflammation and eosinophilic infiltration are prominent pathologic features of chronic rhinosinusitis with nasal polyps (CRSwNP). The purpose of the present study was to determine the roles of Tregs in controlling Type 2 inflammation and inhibiting eosinophilic infiltration in CRSwNP. 134 nasal polyps, 67 ostiomeatal complex from CRS, and 62 normal nasal tissues from controls were collected to study the enumeration and function of Tregs cells and the expressions of cytokine profiles via immunofluorescence staining, flow cytometry, qRT-PCR, ELISA, and/or H&E staining. The effects of Tregs on Th2 and Th17 cells were determined in an eosinophilic chronic sinusitis (ECRS) mice model. It was confirmed that the CRSwNP displayed the feature of Th2 and Th17 cells-mediated inflammation, accompanying by an increased level of eosinophilic infiltration and the eosinophil cationic protein, with a decreased frequency of Treg cells. Furthermore, the percentages of CD4+CD25+CD127lowTreg and CD4+CD25+Foxp3+Treg were only decreased in the polyps of CRSwNP but not in the paired peripheral blood. The CRSwNP possessed the decreased Nrp1+Tregs, Helios+Treg, and low TGF-β and IL-10 expressions in Tregs. The ECRS mice showed similar inflammatory characteristics to CRSwNP patients. The adoptive transfer of CD4+CD25+Foxp3+ Treg cells significantly decreased the inflammatory cytokines, eosinophilic chemotactic factors in the mucosa of the ECRS mice without alteration of the immune balance in the peripheral blood and spleen. In conclusion, CRSwNP showed high Th2 and Th17 inflammation and defective Tregs. The iTreg may correct the imbalance between immune tolerance and effect via limiting the eosinophil recruitment of mucosa in CRSwNP.


Introduction
Darmstadt, Germany). After blocking with 5% BSA for 1 h at room temperature, the membranes 139 were incubated overnight at 4°C with rabbit anti-human Foxp3 antibody (cst) or GAPDH 140 antibodies (proteintech, Chicago, United States) overnight and then incubated with 141 HRP-conjugated secondary antibodies, the membranes were visualized by using the ECL 142 reaction (advansta, California, USA) with Image Reader. The FluorChem M System (protein 143 simple, Alpha Innotech Corp.) was used to visualize the protein bands and capture images of 144 the gels. Image J software was used to analyze and quantify the density of the Western blot 145 bands.

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Immunofluorescence 148 Frozen slice sections of the tissues stored in −80℃ were immersed in acetone for 10 min, 149 following which the sections were allowed to warm up to room temperature, and then washed   respectively. On day 103, all mice were sacrificed by CO 2 asphyxiation, followed by cervical 184 dislocation. Nasal lavage fluid (NLF) and peripheral blood were collected from half mice of each 185 group for the further analysis. Another half of mice in each group were used to collect nasal 186 sinuses. The nasal sinuses were decalcified, embedded in paraffin, and sectioned. Histological 6 examination was performed as described above. The spleens were processed to isolate single 188 cell suspensions for flow cytometry analysis. 189 190 iTreg and control cell preparation 191 Naïve splenic CD4 + CD25 + CD44 low CD62L high cells were stimulated with anti-CD3/CD28 beads 192 (Invitrogen, Carlsbad, CA) at a bead/T cell ratio of 1:5 in the presence of IL-2 (20 U/ml, R&D 193 system) and TGF-β (2 ng/ml, R&D system) for the generation of Foxp3 + Tregs for injections, or 194 only in the presence of IL-2 (20 U/ml, R&D system) for the generation of control cells, as 195 previous research described [28,29].    or Student t-tests were used for between-group comparisons for unpaired data. P <0.05 was 244 accepted as statistically significant.

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In our previous study, we reported that the Chinese form of CRSwNP was primarily 249 characterized by the increased eosinophilic infiltration and edema, and with significantly   293 To further determine if the decreased quantity of Treg cells in CRSwNP could be due either to 294 local inflammation of the nasal mucosa or to systemic inflammatory responses, the differences 9 in the percentages of CD4 + CD25 + Foxp3 + T cells and CD4 + CD25 + CD127 low T cells were 296 compared between diseased polyps and paired peripheral blood of the same CRSwNP 297 patients. As shown in figure 4, the percentages of CD4 + CD25 + Foxp3 + T cells and 298 CD4 + CD25 + CD127 low T cells in polyps of nearly all patients with CRSwNP were significantly 299 lower than that in paired peripheral blood of the same patients (P = 0.027 for 300 CD4 + CD25 + Foxp3 + T cells, P=0.008 for CD4 + CD25 + CD127 low T cells). Even if the difference of

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As iTreg subset is more stable in the inflammatory conditions and more stable in numbers 326 gained [38-40], we have used this Treg subset to determine the effect on ECRS animal model.

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As shown in Figure 6-B and C, percentages of the eosinophils was significantly greater among 328 the total inflammatory cells in the ECRS mice than in those in the control mucosa (P<0.001).

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Nonetheless, the intravenous injection of iTreg subset has significantly decreased the 330 eosinophil counts in the mucosa of ECRS mice, suggesting that iTreg infusion has attenuated 331 the eosinophilic infiltration and weakened the mucosal inflammation.

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The intravenous injection of iTreg released the mucosal type 2/17 bias inflammation 334 We also evaluated whether Treg infusion can control Th2 anf Th17 immune response in the 335 ECRS murine model. Th2 and Th17 relative cytokines, as well as eosinophilic chemotactic CD4 + CD25 + Foxp3 + Tregs are significantly low [48]. Our study found out that Chinese CRSwNP 386 is associated with a down regulation of CD4 + CD25 + Foxp3 + Tregs, CD4 + CD25 + CD127 low , and  increased percentages of CD4 + CD25 + Foxp3 + Tregs [48]. All above studies have demonstrated 413 that increased Treg cells would be a useful method to control the type 2 cytokines-mediated 414 chronic inflammation.

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In our previous studies, we reported that the TGF-β-induced CD4 + Foxp3 + T cells (induced     Clinical Science. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version is available at https://doi.org/10.1042/CS20190388