Increased Phenotypic Differentiation and Reduced Corticosteroid Sensitivity

Lo 11 Increased phenotypic differentiation and reduced corticosteroid sensitivity 2 of fibrocytes in severe asthma34 Chun-Yu Lo MD *#$, Charalambos Michaeloudes PhD *$, Pankaj K Bhavsar PhD *, Chien-5 Da Huang MD#, Chun-Hua Wang MD#, Han-Pin Kuo MD# and Kian Fan Chung MD*67 8 * Airway Disease Section, National Heart and Lung Institute, 9 Imperial College London and Biomedical Research Unit, Royal Brompton Hospital, London, UK.10 # Department of Thoracic Medicine, Chang Gung Medical Foundation, Chang Gung11 University College of Medicine, Taipei, Taiwan12 $ These authors have contributed equally to this work.1314 Corresponding author: Professor Kian Fan Chung, National Heart and Lung Institute,15 Airway Disease Section, Guy Scadding Building, Dovehouse Street,16 London SW3 6LY; e-mail: [email protected]; Telephone: 0044 20 759479591718 This work was supported by the Chang Gung Medical Research Project (CMRP) grant19 G3A0872 and the National Institute of Health Research Respiratory Disease Biomedical20 Research Unit at the Royal Brompton Hospital and Harefield Foundation NHS Trust and21 Imperial College London. Lo 222 ABSTRACT23 BACKGROUND: Patients with severe asthma are less responsive to corticosteroid therapy24 and show increased airway remodelling. The mesenchymal progenitors, fibrocytes, may be25 involved in the remodelling of asthmatic airways. We propose that fibrocytes in severe26 asthma are different from those from non-severe asthma. OBJECTIVE: To examine the27 survival, myofibroblastic differentiation and C-C chemokine receptor 7 (CCR7) expression in28 blood fibrocytes from patients with severe and non-severe asthma, and study the effect of29 corticosteroids on fibrocyte function. 30 METHODS: The non-adherent non-T (NANT) cell fraction of blood mononuclear cells was31 isolated from healthy subjects and patients with non-severe and severe asthma. Total and32 differentiating fibrocytes were identified by their expression of CD45, collagen I and α-33 smooth muscle actin using flow cytometry. The expression of CCR7 and of the34 glucocorticoid receptor (GR) was measured by flow cytometry.35 RESULTS: Increased numbers of circulating fibrocytes, with greater myofibroblastic36 differentiation potential were observed in patients with severe asthma. Dexamethasone37 induced apoptosis, leading to reduction in the number of cultured fibrocytes and total NANT38 cells from healthy subjects and patients with non-severe asthma but not from patients with39 severe asthma. Dexamethasone reduced CCR7 expression in fibrocytes from patients with40 non-severe asthma but not from patients with severe asthma. GR expression was attenuated in41 fibrocytes from patients with severe asthma. 42 CONCLUSIONS: Patients with severe asthma have elevated numbers of circulating43 fibrocytes that show enhanced myofibroblastic differentiation and that are less responsive to44 the effects of corticosteroids. 4546 KEY MESSAGES Lo 347  Patients with severe asthma have a higher number of circulating fibrocytes which48 have a higher capacity to undergo myofibroblastic differentiation in culture.49  Fibrocytes of patients with severe asthma are more resistant to the induction of50 apoptosis and to the inhibition of CCR7 expression by corticosteroids.51 CAPSULE SUMMARY52 Patients with severe asthma have increased circulating fibrocytes which show a propensity to53 differentiate into myofibroblasts and are relatively corticosteroid-insensitive. Altered54 fibrocyte function may contribute to the exaggerated airway remodelling observed in these55 patients.56 KEY WORDS57 Fibrocytes, asthma, corticosteroids, remodelling, myofibroblasts, glucocorticoid receptor,58 CCR7, non-adherent non-T cells, PBMCs59 ABBREVIATIONS 60 ASM, Airway smooth muscle; α-SMA, α-smooth muscle actin; PBMCs, peripheral blood61 mononuclear cells; GR, glucocorticoid receptor; NANT, Non-adherent non-T cell 6263646566 INTRODUCTION67 The airways in asthma are characterised by airway wall remodeling, including thickening of Lo 468 the airway smooth muscle (ASM) layer and subepithelial fibrosis, which could contribute to69 airflow obstruction (1). ASM layer thickening and subepithelial fibrosis in asthma may result70 from ASM cell (ASMC) hyperplasia and hypertrophy (2;3), and increased numbers of71 myofibroblasts (4) in the airway wall. A proportion of asthmatic patients suffer from severe or72 refractory asthma which is difficult to control despite receiving high doses of inhaled and73 sometimes oral corticosteroids (5). These patients show increased airway wall remodeling,74 with augmented subepithelial fibrosis and ASM thickening, which may contribute to the75 persistent airflow obstruction in these patients (6-8). 76 Fibrocytes are bone marrow-derived circulating mesenchymal progenitor cells that express77 leukocyte markers such as CD34 and CD45 but also mesenchymal markers such as pro-78 collagen and α-smooth muscle actin (α-SMA) (9). Fibrocytes migrate to the lung in response79 to inflammation, and home on to the airway wall where they differentiate into80 myofibroblasts. One mediator of fibrocyte migration to the lungs is the C-C chemokine81 receptor 7 (CCR7) (10;11). CCR7 is expressed on circulating fibrocytes (12), while the expression82 of its ligand, chemokine (C-C) motif ligand (CCL)-19, is increased in the ASM of patients83 with asthma which implicates an important role of CCR7 in the homing of fibrocytes to84 asthmatic airways (13). Fibrocytes are more abundant in the circulation of asthma subjects85 with chronic airflow obstruction, and they have been localised to the ASM compartment in86 the airway wall of patients with severe refractory asthma (12;14). At the tissue site, fibrocytes87 can differentiate into myofibroblasts (15) which mediate subepithelial fibrosis through the88 release of extracellular matrix (ECM) proteins (4) and may also contribute to increased ASM89 mass (16). 90 Corticosteroid insensitivity is a feature of severe asthma. Corticosteroids are less effective in91 suppressing the release of pro-inflammatory cytokines from peripheral blood mononuclear92 cells (PBMCs) (17;18) and alveolar macrophages (19) from patients with severe asthma. ASMCs Lo 593 from patients with severe asthma are also resistant to the anti-proliferative and anti-94 inflammatory effects of corticosteroids (20;21). Moreover, T lymphocytes from patients with95 severe refractory asthma, and PBMCs exposed to interleukin (IL)-17 and IL-23 are resistant96 to the induction of cell cycle arrest and apoptosis by corticosteroids (21-23). However, the effect97 of corticosteroids on fibrocytes from patients with severe asthma is not known. 98 We hypothesized that patients with severe asthma have higher numbers of circulating99 fibrocytes with increased differentiation potential and decreased sensitivity to the effects of100 corticosteroids. We therefore determined the number of total and differentiating fibrocytes in101 the non-adherent non-T (NANT) cell fraction of PBMCs of healthy subjects and patients with102 non-severe and severe asthma, immediately after isolation and also following culture. We103 also compared the expression of the glucocorticoid receptor (GR) in fibrocytes and the effect104 of dexamethasone on the number of fibrocytes and the expression of CCR7 in these cells. The105 effect of dexamethasone on apoptosis was also studied in fibrocytes isolated from the106 adherent fraction of PBMCs.107108109110111112 MATERIALS AND METHODS113 Study population114 Healthy subjects and patients with non-severe and severe asthma were recruited. Subjects Lo 6115 with FEV1 reversibility of less than 12% or with a provocative concentration of methacholine116 causing a 20% fall in FEV1 (PC20) of less than 8 mg/mL were diagnosed as asthmatics.117 Severe asthma was defined according to the ATS guidelines for refractory asthma, with the118 presence of at least one of two major criteria for corticosteroid usage, and at least two minor119 criteria (24). Patients with non-severe asthma had perfect control of their symptoms using120 inhaled beclomethasone (0-1000 µg/d or equivalent). Current smokers and ex-smokers with121 a smoking history of greater than 5 pack-years were excluded. All patients provided informed122 consent and the study has been approved by the National Research Ethics Service Committee123 London - Chelsea (REC 08/H0708/109).124 Isolation of circulating fibrocytes from the non-adherent non-T (NANT) cell fraction125 Non-adherent non-T (NANT) cells were isolated from peripheral blood as previously126 described (12). PBMCs were isolated from whole blood using density gradient centrifugation127 with Ficoll-PaqueTM PLUS (GE HealthCare, Uppsala, Sweden). Adherent cells were removed128 by adherence and the non-adherent cell fraction was depleted of T cells using magnetic beads129 coated with anti-CD3 mAb (Miltenyi Biotec, Auburn, California). The resulting NANT cells130 were cultured in Iscove Modified Dulbecco medium (IMDM; Sigma-Aldrich, Ayrshire, UK)131 containing 30% FBS and 1% BSA at 37 °C and 5% CO2 for the required time periods in the132 presence or absence of the required treatments. The number of viable NANT cells was133 determined by Trypan blue staining and haemocytometer counting. 134 Fibrocytes were identified in freshly isolated or cultured NANT cells as CD45+/collagen I+135 cells, whilst differentiating fibrocytes as α-SMA+ cells, using flow cytometry. NANT cells136 were fixed using 0.5% paraformaldehyde, washed twice with PBS and permeabilised using137 BD FACS™ Permeabilizing Solution 2 (BD Biosciences, San Jose, California). Cell pellets138 were incubated with mouse anti-human collagen I antibody (Millipore Corporation, Lo 7139 Temecula, California) followed by a fluorescein isothiocyanate (FITC)-conjugated anti-140 mouse secondary antibody (DAKO A/S, Glostrup, Denmark). Cell pellets were then141 incubated with an allophycocyanin (APC)-conjugated mouse-anti-human CD45 antibody142 (BD Biosciences). In some experiments cells were also stained using a phycoerythrin (PE)-143 conjugated mouse anti-human CCR7 antibody (BD Biosciences). Stained cells were analysed144 using a BD FACSCantoTM II flow cytometer. The relevant isotype antibody controls were145 used for every flow cytometry experiment.146 Isolation of fibrocytes from adherent PBMCs 147 In some experiments fibrocytes isolated from the adherent fraction of PBMCs were used.148 Briefly, PBMCs were cultured in Dulbecco's Modified Eagle's medium (DMEM) (Sigma-149 Aldrich) supplemented with 10% FBS for 3 days. Non-adherent cells were then removed and150 adherent cells were cultured for a further 3 days in the presence of the required treatments.151 Viable cells were counted and the percentage of fibrocytes was determined as described152 above.153 Determination of GR expression in fibrocytes154 NANT cells were fixed, stained with an APC-conjugated mouse anti-human CD45 antibody155 (BD Biosciences) and permeabilised as described above. Cell pellets were then incubated156 with a FITC-conjugated mouse anti-human collagen I antibody (Millipore), and a rabbit-anti-157 human GR antibody (Abcam, MA, USA) followed by a PE-conjugated goat-anti-rabbit158 antibody (Abcam). Stained cells were analysed by flow cytometry as described above.159 Determination of apoptotic NANT cells using Annexin V and propidium iodide (PI)160 staining161 Apoptosis of NANT cells was analysed by Annexin V and propidium iodide (PI) staining Lo 8162 using the FITC Annexin V/Dead cell apoptosis kit (Invitrogen) according to the163 manufacturer’s instructions. Briefly, NANT cells were treated as required, washed in PBS164 and then were incubated with FITC-conjugated annexin V and PI, and analysed by flow165 cytometry. Annexin V-/PI- were considered live cells, Annexin V+/PI- as apoptotic and166 annexin V+/PI+ as late apoptotic cells. 167 Statistical analysis168 Data are presented as mean ± standard error of the mean (SEM). Statistical analysis was169 carried out using the GraphPad Prism v.5 software package (GraphPad Prism Software Inc,170 San Diego, CA). Results were analysed using the Friedman test for intra-group comparisons,171 and the Kruskal-Wallis test for comparisons between healthy and non-severe and severe172 asthmatic groups, followed by Dunns post test. Correlations between parameters were173 determined by Spearman’s rank correlation. p<0.05 was considered as statistically174 significant.175176 RESULTS177 Increased circulating fibrocytes in patients with severe asthma178 Immediately after isolation there was no significant difference in the number of NANT cells179 per mL of blood between the three groups (See Figure E1 in the Online Repository).180 However, the NANT cells of severe asthma patients contained a higher percentage of181 fibrocytes compared to that of non-severe asthma patients and healthy subjects, whilst the182 percentage of circulating fibrocytes in non-severe asthma patients was higher than in healthy183 subjects (Figure 1A and B). These differences were thus reflected in the absolute number of184 fibrocytes per mL of blood which was increased in severe asthmatics compared to non-severe185 asthmatics and healthy subjects, but also in non-severe asthmatics compared to healthy Lo 9186 subjects (Figure 1C). The number of circulating fibrocytes in severe asthmatics who were on187 oral prednisolone was not significantly different from that of patients who were not (See188 Figure E2 in the Online Repository), suggesting that systemic corticosteroid therapy did not189 affect the number of circulating fibrocytes. Moreover, the number of fibrocytes per mL of190 blood showed an inverse correlation with the pre-bronchodilator FEV1% (Figure 1D) and191 FEV1/FVC ratio (Figure 1E). 192 Increased differentiation of fibrocytes from patients with severe asthma193 We observed an increase in the number of fibrocytes in the NANT cells from healthy subjects194 and patients with non-severe asthma following culture, peaking after 3 days in culture, whilst195 the number of fibrocytes from patients with severe asthma did not increase (Figure 2A). We196 employed single α-SMA staining to identify differentiating fibrocytes as we have established197 that the majority cells in the NANT cell population expressing α-SMA are fibrocytes (data198 not shown). The number of differentiating fibrocytes in the NANT cells of healthy subjects199 increased with time, peaking after 3 days (Figure 2B). The changes in α-SMA-positive cells200 were followed by an increase in adherent “spindle-shaped” cells, which peaked after 10 days201 in culture, indicating that fibrocytes can fully differentiate into myofibroblasts-like cells (See202 Figure E3 in the Online Repository). We observed an increase in the number of203 differentiating fibrocytes in the NANT cells of all groups, which also peaked after 3 days in204 culture (Figure 2B). However, the number of differentiating fibrocytes in the NANT cells205 from patients with severe asthma was higher compared to healthy subjects and patients with206 non-severe asthma at day 3 (Figure 2B). 207 Effect of corticosteroids on apoptosis of NANT cells and fibrocytes 208 Dexamethasone (10-8-10-6 M) treatment for 3 days led to a concentration-dependent reduction209 in the number of fibrocytes and differentiating fibrocytes from healthy subjects and patients Lo 10210 with non-severe asthma, but had no effect on those from patients with severe asthma (Figures211 3A and 3B). Dexamethasone (10-8-10-6 M) also reduced the number of the total NANT cells212 from healthy subjects and patients with non-severe asthma but not those from patients with213 severe asthma (Figure 3C). 214 We determined the percentage of apoptotic NANT cells both at baseline and after215 dexamethasone treatment. Immediately after isolation, the percentage of live and apoptotic216 cells was similar in all groups (Figure 3D). After 3 days in culture, untreated NANT cells217 from patients with severe asthma contained a higher percentage of live cells and a lower218 percentage of early apoptotic cells compared to healthy subjects, suggesting increased219 survival of severe asthmatic NANT cells (Figures 3E). Dexamethasone caused a reduction in220 the percentage of live cells whilst increasing the percentage of early and late apoptotic cells221 in NANT cells from healthy subjects (Figure 3F) and patients with non-severe asthma (Figure222 3G). However, dexamethasone did not modulate the percentage of live and apoptotic NANT223 cells from patients with severe asthma (Figure 3H). Similarly, when the data was expressed224 as a total percentage of apoptotic cells, NANT cells from patients with severe asthma showed225 a higher percentage of live, and a lower percentage of apoptotic, cells compared to those from226 healthy subjects (See Table E1 in the Online Repository). Moreover, dexamethasone caused a227 significant decrease in the percentage of total apoptotic cells from healthy subjects and228 patients with non-severe asthma but had no effect on those from patients with severe asthma229 (See Table E1 in the Online Repository). 230 Dexamethasone (10-7 M)-induced reduction in NANT cell (Figure 4A), fibrocyte (Figure 4B)231 and differentiating fibrocyte (Figure 4C) numbers was prevented by the GR antagonist232 RU486 (10-6-10-5 M), confirming that this effect is GR-mediated.233 Corticosteroid insensitivity of fibrocytes on CCR7 expression in severe asthma Lo 11234 No significant difference was observed in the baseline percentage of CCR7-positive235 fibrocytes between the three groups (Figure 5A-B). However, dexamethasone (10-8-10-6 M)236 treatment for 3 days led to a concentration-dependent decrease in the percentage of CCR7-237 positive fibrocytes (Figure 5A and C) and the CCR7 MFI ratio (Figure 5D) in NANT cells238 from patients with non-severe asthma, but had no significant effect on those from patients239 with severe asthma. Thus, the expression of CCR7 on the fibrocytes of patients with severe240 asthma is resistant to the inhibitory effect of dexamethasone.241 GR expression in fibrocytes in severe asthma 242 We compared the expression of GR in fibrocytes in freshly isolated NANT cells from healthy243 subjects and patients with non-severe and severe asthma (Figure 6A). Both the percentage of244 GR-positive fibrocytes (Figure 6B) and the GR MFI ratio (Figure 6C) were lower in patients245 with severe asthma compared to healthy subjects and patients with non-severe asthma. The246 percentage of GR-positive cells and GR MFI ratio were also reduced in the whole NANT cell247 isolated from patients with severe asthma compared to healthy subjects and patients with248 non-severe asthma (See Figure E4 in the Online Repository).249250251 Effect of dexamethasone on fibrocytes isolated from adherent PBMCs252 The percentage of fibrocytes and differentiating fibrocytes in adherent PBMCs increased in a253 time-dependent manner peaking after 10 days in culture (See Figure E5 in the Online254 Repository). Treatment with dexamethasone (10-7 M) for 72 hrs led to a decrease in the255 number of fibrocytes and differentiating fibrocytes from healthy subjects, and this effect was256 prevented by RU486 (10-6 M) (Figures 7A and 7B). Dexamethasone (10-7 M) also reduced the257 number of fibrocytes and differentiating fibrocytes from patients with non-severe asthma but258 had no significant effect on those from patients with severe asthma (Figures 7C and 7D). Lo 12259 Thus, fibrocytes isolated from the adherent fraction of PBMCs of severe asthmatic patients260 are also resistant to corticosteroid-induced apoptosis.261262263264265266267268269270271272 DISCUSSION273 We have demonstrated that, immediately after isolation, there is a higher number of274 fibrocytes in the circulating blood of patients with severe asthma compared to healthy275 subjects and patients with non-severe asthma. Furthermore, we showed that, when placed in276 culture, fibrocytes from patients with severe asthma have a greater capacity to differentiate277 into myofibroblasts compared to fibrocytes from healthy subjects and patients with non-278 severe asthma. Dexamethasone decreased the number of NANT cells, including fibrocytes,279 from healthy subjects and patients with non-severe asthma but had no effect on those from Lo 13280 patients with severe asthma. In line with these findings, dexamethasone reduced the281 percentage of live NANT cells, and increased the percentage of apoptotic NANT cells from282 healthy subjects and patients with non-severe asthma but had no effect on those from patients283 with severe asthma. Fibrocytes from patients with severe asthma were also resistant to the284 inhibition of CCR7 expression by dexamethasone and showed reduced GR expression285 compared to those from healthy subjects and patients with non-severe asthma. Thus, patients286 with severe asthma have a higher number of circulating fibrocytes that have a greater287 capacity to differentiate into myofibroblasts while showing relative corticosteroid288 insensitivity both in terms of apoptotic response and suppression of CCR7 expression. 289 The airway wall in severe asthma is characterized by increased ASM thickening and sub-290 epithelial fibrosis, features of a remodelling process (6-8). We show that fibrocytes are more291 abundant in the circulation of patients with severe asthma and have an increased capacity to292 differentiate into myofibroblasts compared to healthy subjects and patients with non-severe293 asthma. Our data are in line with findings by Saunders et al showing increased numbers of α-294 SMA-positive fibrocytes in the lamina propria of patients with severe refractory asthma and295 in the ASM bundles of patients with asthma of all severities, suggesting that fibrocytes can296 migrate to the airway wall of these patients and differentiate into myofibroblasts (14).297 Myofibroblasts exhibit a phenotype between fibroblasts and smooth muscle cells that298 involves secretion of ECM proteins (25) but also expression of smooth muscle-specific299 proteins (26). The number of myofibroblasts in the airways of patients with asthma was found300 to be correlated with the extent of subepithelial fibrosis (4). Myofibroblasts have also been301 detected in the ASM bundles of patients with asthma, therefore possibly contributing to the302 increased ASM mass (16). Thus, patients with severe asthma may have a greater pool of303 circulating fibrocytes which can readily differentiate into myofibroblasts upon migration to304 the airway wall, contributing to the remodelling process. Lo 14305 Patients with severe asthma show exaggerated airway remodelling despite being on high306 doses of inhaled and sometimes oral corticosteroid treatment (6-8). We, therefore, determined307 the effect of corticosteroids on the function of fibrocytes isolated from these patients. Our308 data show a reduction in the number of fibrocytes and differentiating fibrocytes from healthy309 subjects and patients with non-severe asthma in response to exposure to dexamethasone,310 which was prevented by the GR antagonist, RU486. Moreover, dexamethasone reduced the311 number of total NANT cells whilst increasing the percentage of apoptotic NANT cells,312 implicating GR-mediated apoptosis in this effect. Hayashi et al have also recently reported313 dexamethasone-induced inhibition of the proliferation of circulating fibrocytes of healthy314 subjects (27). Dexamethasone also led to a reduction in the expression of CCR7 in the315 fibrocytes from patients with non-severe asthma, in line with reports in dendritic cells (28;29),316 suggesting that corticosteroids may have an inhibitory effect on the migration of fibrocytes.317 Intriguingly, dexamethasone had no effect on both the number or on the expression of CCR7318 in fibrocytes from patients with severe asthma indicating that these cells show relative319 corticosteroid insensitivity. This may result from the lower GR expression levels we observed320 in fibrocytes from severe asthma. Indeed, PBMCs from patients with asthma treated with IL-321 2 and IL-4 show reduced GR-alpha expression accompanied by corticosteroid insensitivity,322 including resistance to dexamethasone-induced apoptosis (23). However, other mechanisms323 such as reduced histone deacetylase activity (17) and p38 mitogen activated protein kinase-324 dependent GR phosphorylation (18-20;30) may also be involved. Dexamethasone also failed to325 induce apoptosis and reduce the number of total NANT cells from patients with severe326 asthma. NANT cells from patients with severe asthma also exhibited lower levels of GR327 expression compared to healthy subjects and patients with severe asthma. Thus, the relative328 corticosteroid insensitivity we observed in fibrocytes from patients with severe asthma also329 extends to non-fibrocyte cells. Nonetheless, as we have shown that NANT cells from patients Lo 15330 with severe asthma show increased survival, we cannot exclude the possibility that the failure331 of dexamethasone to reduce the number these cells is a result of an inherent resistance to332 apoptosis.333 We have previously demonstrated relative corticosteroid insensitivity in PBMCs (17), alveolar334 macrophages (19) as well as ASMCs (20) from patients with severe asthma. We now extend this335 observation to show that in patients with severe asthma, circulating progenitor cells such as336 fibrocytes may also exhibit an altered response to corticosteroids. This finding may have337 important implications in understanding the mechanisms underlying the increased338 remodelling observed in these patients. 339 The most commonly described method of isolating fibrocytes from blood uses the adherent340 fraction of PBMCs (31;32). However, fibrocytes are also abundant in the non-adherent fraction341 of PBMCs (12;33). We found that fibrocytes isolated from the adherent fraction of PBMCs of342 patients with severe asthma were also resistant to the pro-apoptotic effect of dexamethasone,343 suggesting that both adherent and non-adherent fibrocyte populations show the same pattern344 of response to corticosteroids in vitro.345 A limitation of our study is that it does not provide direct evidence for the role of fibrocyte-346 derived myofibroblasts in the development of airway remodelling in severe asthma. A study347 involving tracking fluorescence-labeled fibrocytes in a mouse model of allergic asthma has348 shown recruitment of fibrocytes from the circulation to the bronchial tissue and349 differentiation into myofibroblasts in response to allergen exposure (15). Similar studies in350 animal models of severe asthma may provide a clearer picture of the involvement of351 fibrocytes in the development of airway remodelling in this disease.352 Fibrocytes are potentially important cells involved in the airway wall remodeling in asthma353 (10). Our study demonstrates that patients with severe asthma have elevated numbers of Lo 16354 circulating fibrocytes which show an aberrant phenotype involving a heightened ability to355 differentiate into myofibroblasts and relative resistance to the effects of corticosteroids.356 Fibrocytes could thus play a central role in the development and persistence of airway357 remodelling in severe asthma and could be an important therapeutic target. 358 359 ACKNOWLEDGEMENTS 360361 We thank João Pedro Carvalho da Purificacao Rocha for recruiting the subjects and362 Kirandeep K. Chana, Po-Jui Chang, Chih-Ming Weng, and Kang-Yun Lee for their technical363 help and advice.364365366367368 References369370 (1) Jeffery PK. Remodeling and inflammation of bronchi in asthma and chronic 371 obstructive pulmonary disease. Proc Am Thorac Soc 2004; 1(3):176-83.372 (2) James AL, Elliot JG, Jones RL, Carroll ML, Mauad T, Bai TR et al. Airway 373 smooth muscle hypertrophy and hyperplasia in asthma. Am J Respir Crit Care Med 2012; 374 185(10):1058-64.375 (3) Woodruff PG, Dolganov GM, Ferrando RE, Donnelly S, Hays SR, Solberg 376 OD et al. Hyperplasia of smooth muscle in mild to moderate asthma without changes in cell 377 size or gene expression. Am J Respir Crit Care Med 2004; 169(9):1001-6.378 (4) Brewster CE, Howarth PH, Djukanovic R, Wilson J, Holgate ST, Roche WR. 379 Myofibroblasts and subepithelial fibrosis in bronchial asthma. Am J Respir Cell Mol Biol 380 1990; 3(5):507-11.381 (5) Gibeon D, Chung KF. The investigation of severe asthma to define 382 phenotypes. Clin Exp Allergy 2012; 42(5):678-92.383 (6) Benayoun L, Druilhe A, Dombret MC, Aubier M, Pretolani M. Airway 384 structural alterations selectively associated with severe asthma. Am J Respir Crit Care Med Lo 17385 2003; 167(10):1360-8.386 (7) Bumbacea D, Campbell D, Nguyen L, Carr D, Barnes PJ, Robinson D et al. 387 Parameters associated with persistent airflow obstruction in chronic severe asthma. Eur 388 Respir J 2004; 24(1):122-8.389 (8) Macedo P, Hew M, Torrego A, Jouneau S, Oates T, Durham A et al. 390 Inflammatory biomarkers in airways of patients with severe asthma compared with non- 391 severe asthma. Clin Exp Allergy 2009; 39(11):1668-76.392 (9) Peng H, Herzog EL. Fibrocytes: emerging effector cells in chronic 393 inflammation. Curr Opin Pharmacol 2012; 12(4):491-6.394 (10) Gomperts BN, Strieter RM. Fibrocytes in lung disease. J Leukoc Biol 2007; 395 82(3):449-56.396 (11) Phillips RJ, Burdick MD, Hong K, Lutz MA, Murray LA, Xue YY et al. 397 Circulating fibrocytes traffic to the lungs in response to CXCL12 and mediate fibrosis. J Clin 398 Invest 2004; 114(3):438-46.399 (12) Wang CH, Huang CD, Lin HC, Lee KY, Lin SM, Liu CY et al. Increased 400 circulating fibrocytes in asthma with chronic airflow obstruction. Am J Respir Crit Care Med 401 2008; 178(6):583-91.402 (13) Kaur D, Saunders R, Berger P, Siddiqui S, Woodman L, Wardlaw A et al. 403 Airway smooth muscle and mast cell-derived CC chemokine ligand 19 mediate airway 404 smooth muscle migration in asthma. Am J Respir Crit Care Med 2006; 174(11):1179-88.405 (14) Saunders R, Siddiqui S, Kaur D, Doe C, Sutcliffe A, Hollins F et al. Fibrocyte 406 localization to the airway smooth muscle is a feature of asthma. J Allergy Clin Immunol 407 2009; 123(2):376-84.408 (15) Schmidt M, Sun G, Stacey MA, Mori L, Mattoli S. Identification of 409 circulating fibrocytes as precursors of bronchial myofibroblasts in asthma. J Immunol 2003; 410 171(1):380-9.411 (16) Begueret H, Berger P, Vernejoux JM, Dubuisson L, Marthan R, Tunon-de- 412 Lara JM. Inflammation of bronchial smooth muscle in allergic asthma. Thorax 2007; 62(1):8- 413 15.414 (17) Hew M, Bhavsar P, Torrego A, Meah S, Khorasani N, Barnes PJ et al. 415 Relative corticosteroid insensitivity of peripheral blood mononuclear cells in severe asthma. 416 Am J Respir Crit Care Med 2006; 174(2):134-41.417 (18) Mercado N, Hakim A, Kobayashi Y, Meah S, Usmani OS, Chung KF et al. 418 Restoration of corticosteroid sensitivity by p38 mitogen activated protein kinase inhibition in 419 peripheral blood mononuclear cells from severe asthma. PLoS One 2012; 7(7):e41582.420 (19) Bhavsar P, Hew M, Khorasani N, Torrego A, Barnes PJ, Adcock I et al. 421 Relative corticosteroid insensitivity of alveolar macrophages in severe asthma compared with 422 non-severe asthma. Thorax 2008; 63(9):784-90. Lo 18423 (20) Chang PJ, Bhavsar PK, Michaeloudes C, Khorasani N, Chung KF. 424 Corticosteroid insensitivity of chemokine expression in airway smooth muscle of patients 425 with severe asthma. J Allergy Clin Immunol 2012; 130(4):877-85.426 (21) Roth M, Johnson PR, Borger P, Bihl MP, Rudiger JJ, King GG et al. 427 Dysfunctional interaction of C/EBPalpha and the glucocorticoid receptor in asthmatic 428 bronchial smooth-muscle cells. N Engl J Med 2004; 351(6):560-74.429 (22) Corrigan CJ, Brown PH, Barnes NC, Tsai JJ, Frew AJ, Kay AB. 430 Glucocorticoid resistance in chronic asthma. Peripheral blood T lymphocyte activation and 431 comparison of the T lymphocyte inhibitory effects of glucocorticoids and cyclosporin A. Am 432 Rev Respir Dis 1991; 144(5):1026-32.433 (23) Vazquez-Tello A, Halwani R, Hamid Q, Al-Muhsen S. Glucocorticoid 434 receptor-beta up-regulation and steroid resistance induction by IL-17 and IL-23 cytokine 435 stimulation in peripheral mononuclear cells. J Clin Immunol 2013; 33(2):466-78.436 (24) Proceedings of the ATS workshop on refractory asthma: current 437 understanding, recommendations, and unanswered questions. American Thoracic Society. 438 Am J Respir Crit Care Med 2000; 162(6):2341-51.439 (25) Sugiura H, Ichikawa T, Koarai A, Yanagisawa S, Minakata Y, Matsunaga K et 440 al. Activation of Toll-like receptor 3 augments myofibroblast differentiation. Am J Respir 441 Cell Mol Biol 2009; 40(6):654-62.442 (26) Gizycki MJ, Adelroth E, Rogers AV, O'Byrne PM, Jeffery PK. Myofibroblast 443 involvement in the allergen-induced late response in mild atopic asthma. Am J Respir Cell 444 Mol Biol 1997; 16(6):664-73.445 (27) Hayashi H, Kawakita A, Okazaki S, Murai H, Yasutomi M, Ohshima Y. IL-33 446 enhanced the proliferation and constitutive production of IL-13 and IL-5 by fibrocytes. 447 Biomed Res Int 2014; 2014:738625.448 (28) Larange A, Antonios D, Pallardy M, Kerdine-Romer S. Glucocorticoids 449 inhibit dendritic cell maturation induced by Toll-like receptor 7 and Toll-like receptor 8. J 450 Leukoc Biol 2012; 91(1):105-17.451 (29) Vizzardelli C, Pavelka N, Luchini A, Zanoni I, Bendickson L, Pelizzola M et 452 al. Effects of dexamethazone on LPS-induced activationand migration of mouse dendritic 453 cells revealed by a genome-wide transcriptional analysis. Eur J Immunol 2006; 36(6):1504- 454 15.455 (30) Irusen E, Matthews JG, Takahashi A, Barnes PJ, Chung KF, Adcock IM. p38 456 Mitogen-activated protein kinase-induced glucocorticoid receptor phosphorylation reduces its 457 activity: role in steroid-insensitive asthma. J Allergy Clin Immunol 2002; 109(4):649-57.458 (31) Chesney J, Metz C, Stavitsky AB, Bacher M, Bucala R. Regulated production 459 of type I collagen and inflammatory cytokines by peripheral blood fibrocytes. J Immunol 460 1998; 160(1):419-25.461 (32) Yang L, Scott PG, Giuffre J, Shankowsky HA, Ghahary A, Tredget EE. 462 Peripheral blood fibrocytes from burn patients: identification and quantification of fibrocytes Lo 19463 in adherent cells cultured from peripheral blood mononuclear cells. Lab Invest 2002; 464 82(9):1183-92.465 (33) Wang CH, Huang CD, Lin HC, Huang TT, Lee KY, Lo YL et al. Increased 466 activation of fibrocytes in patients with chronic obstructive asthma through an epidermal 467 growth factor receptor-dependent pathway. J Allergy Clin Immunol 2012; 129(5):1367-76. 468 469470471472473474475476477478479 FIGURE LEGENDS480 Figure 1. Number of circulating fibrocytes in freshly isolated NANT cells from healthy481 subjects and patients with non-severe and severe asthma. Representative flow cytometry482 scatter plots from one experiment are shown (A). (A-C) The percentage (B) and number (C)483 of fibrocytes (Col I+/CD45+ cells) in freshly isolated NANT cells from healthy subjects (H;484 n=18) and patients with non-severe (NSA; n=18) and severe asthma (SA; n=38) was485 determined. Horizontal lines represent the median for each group. (D-E) Correlation of486 fibrocyte numbers with FEV1 % predicted (D) and FEV1/FVC ratio (E) was determined using487 Spearman’s rank correlation. * p<0.05, ** p<0.01, *** p<0.001. RS: Spearman’s rank488 correlation.489 Lo 20490 Figure 2: Number of fibrocytes and differentiating fibrocytes in cultured NANT cells491 from healthy subjects and patients with non-severe and severe asthma. The number of492 fibrocytes (Col I+/CD45+ cells; A) and differentiated fibrocytes (α-SMA+ cells; B) were493 determined in NANT cells from healthy subjects (n=5-18) and patients with non-severe494 (n=4-18) and severe asthma (n=5-38) after 3-14 days in culture. Bars represent mean ± SEM.495 * p < 0.05, ** p < 0.01, *** p < 0.001 vs day 0 for each patient group. ## p < 0.01 vs healthy496 day 3. $$ p < 0.01 vs non-severe asthma day 3.497 498 Figure 3: Effect of dexamethasone on fibrocyte and differentiating fibrocyte number.499 (A-C) NANT cells from healthy subjects (n=9) and patients with non-severe (n=10) and500 severe asthma (n=12) were cultured for 3 days in the presence or absence of dexamethasone501 (10-8-10-6M). The number of fibrocytes (Col I+/CD45+ cells; A), differentiating fibrocytes (α-502 SMA+ cells; B) and total NANT cells (C) was determined. Data are expressed as fold change503 compared to untreated control. (D-E) The percentage of live, and early and late apoptotic504 NANT cells from healthy subjects (H; n=6) and patients with non-severe (NSA; n=6) and505 severe asthma (SA; n=6) was determined immediately after isolation (D), or after 3 days in506 culture in the absence (E) or presence of dexamethasone (10-7M; F-H). Data points and bars507 represent mean ± SEM. * p<0.05, ** p<0.01, *** p<0.001 vs untreated for each group. #508 p<0.05, ## p<0.01 vs healthy. $ p<0.05, $$ p<0.01 vs non-severe asthma.509510 Figure 4: Effect of GR inhibition on dexamethasone-induced reduction in fibrocyte511 number. NANT cells from healthy subjects were treated with dexamethasone (10-7M) in the512 presence or absence of RU486 (10-6-10-5 M) and total NANT cell (A), fibrocyte (Col513 I+/CD45+ cells; B) and differentiating fibrocyte (α-SMA+ cells; C) numbers were Lo 21514 determined after 3 days. Bars represent mean ± SEM of 6 donors. * p<0.05. 515516 Figure 5: Effect of dexamethasone on CCR7 expression in fibrocytes from patients with517 non-severe and severe asthma. (A) Representative flow cytometry scatter plots from one518 experiment per group are shown. (B) The percentage of CCR7+ fibrocytes (Col519 I+/CD45+/CCR7+ cells) was determined in NANT cells from patients with non-severe520 (NSA; n=10) and severe asthma (SA; n=12) immediately after isolation or after 3 days in521 culture. Horizontal lines represent the median for each group. * p<0.05, ** P<0.01 and ***522 P<0.001. (C-D) The percentage of CCR7+ fibrocytes (C) and the median fluorescence523 intensity (MFI) ratio (D) after treatment with dexamethasone (10-7M) for 3 days was524 determined in the NANT cells of the same patients. Data points represent mean ± SEM. *525 p<0.05, *** p<0.001 vs untreated. # p<0.01 vs non-severe asthma. 526 Figure 6: GR expression in fibrocytes from healthy subjects and patients with non-527 severe and severe asthma. (A) Representative flow cytometry scatter plots from one528 experiment per group are shown. The percentage (B) and median fluorescence intensity529 (MFI) ratios (C) of GR+ fibrocytes (Col I+/CD45+/GR+ cells) were determined in freshly530 isolated NANT cells from healthy subjects (H; n=10) and patients with non-severe (NSA;531 n=8) and severe asthma (SA; n=7). Horizontal lines represent medians. ** p<0.01. 532533 Figure 7. Effect of dexamethasone on the number of fibrocytes derived from adherent534 PBMCs. (A-B) Adherent PBMCs from healthy subjects (n=6) were placed in culture for 3535 days followed by incubation with dexamethasone (10-7 M), in the presence or absence of536 RU486 (10-6 M), for a further 3 days. The number of fibrocytes (Col I+/CD45+ cells; A) and537 differentiating fibrocytes (α-SMA+ cells; B) was determined. (C-D) Adherent PBMCs from Lo 22538 healthy subjects (n=6), and patients with non-severe (n=5) and severe asthma (n=6) were539 placed in culture for 3 days followed by incubation with dexamethasone (10-7 M) for a further540 3 days. The number of fibrocytes (Col I+/CD45+ cells; C) and differentiating fibrocytes (α-541 SMA+ cells; D) was determined. Bars represent mean ± SEM. * p<0.05, ** p<0.01, ***542 p<0.001.543544

Increased Phenotypic Differentiation and Reduced Corticosteroid Sensitivity

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