Increased Phenotypic Differentiation and Reduced Corticosteroid Sensitivity

1 Increased phenotypic differentiation and reduced corticosteroid sensitivity

2 of fibrocytes in severe asthma

4 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*

7 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 Gung

11 University College of Medicine, Taipei, Taiwan

12 $ These authors have contributed equally to this work.

14 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 75947959

18 This work was supported by the Chang Gung Medical Research Project (CMRP) grant

19 G3A0872 and the National Institute of Health Research Respiratory Disease Biomedical

20 Research Unit at the Royal Brompton Hospital and Harefield Foundation NHS Trust and

21 Imperial College London. Lo 2

22 ABSTRACT

23 BACKGROUND: Patients with severe asthma are less responsive to corticosteroid therapy

24 and show increased airway remodelling. The mesenchymal progenitors, fibrocytes, may be

25 involved in the remodelling of asthmatic airways. We propose that fibrocytes in severe

26 asthma are different from those from non-severe asthma. OBJECTIVE: To examine the

27 survival, myofibroblastic differentiation and C-C chemokine receptor 7 (CCR7) expression in

28 blood fibrocytes from patients with severe and non-severe asthma, and study the effect of

29 corticosteroids on fibrocyte function.

30 METHODS: The non-adherent non-T (NANT) cell fraction of blood mononuclear cells was

31 isolated from healthy subjects and patients with non-severe and severe asthma. Total and

32 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 the

34 glucocorticoid receptor (GR) was measured by flow cytometry.

35 RESULTS: Increased numbers of circulating fibrocytes, with greater myofibroblastic

36 differentiation potential were observed in patients with severe asthma. Dexamethasone

37 induced apoptosis, leading to reduction in the number of cultured fibrocytes and total NANT

38 cells from healthy subjects and patients with non-severe asthma but not from patients with

39 severe asthma. Dexamethasone reduced CCR7 expression in fibrocytes from patients with

40 non-severe asthma but not from patients with severe asthma. GR expression was attenuated in

41 fibrocytes from patients with severe asthma.

42 CONCLUSIONS: Patients with severe asthma have elevated numbers of circulating

43 fibrocytes that show enhanced myofibroblastic differentiation and that are less responsive to

44 the effects of corticosteroids.

46 KEY MESSAGES Lo 3

47  Patients with severe asthma have a higher number of circulating fibrocytes which

48 have a higher capacity to undergo myofibroblastic differentiation in culture.

49  Fibrocytes of patients with severe asthma are more resistant to the induction of

50 apoptosis and to the inhibition of CCR7 expression by corticosteroids.

51 CAPSULE SUMMARY

52 Patients with severe asthma have increased circulating fibrocytes which show a propensity to

53 differentiate into myofibroblasts and are relatively corticosteroid-insensitive. Altered

54 fibrocyte function may contribute to the exaggerated airway remodelling observed in these

55 patients.

56 KEY WORDS

57 Fibrocytes, asthma, corticosteroids, remodelling, myofibroblasts, glucocorticoid receptor,

58 CCR7, non-adherent non-T cells, PBMCs

59 ABBREVIATIONS

60 ASM, Airway smooth muscle; α-SMA, α-smooth muscle actin; PBMCs, peripheral blood

61 mononuclear cells; GR, glucocorticoid receptor; NANT, Non-adherent non-T cell

66 INTRODUCTION

67 The airways in asthma are characterised by airway wall remodeling, including thickening of Lo 4

68 the airway smooth muscle (ASM) layer and subepithelial fibrosis, which could contribute to

69 airflow obstruction (1). ASM layer thickening and subepithelial fibrosis in asthma may result

70 from ASM cell (ASMC) hyperplasia and hypertrophy (2;3), and increased numbers of

71 myofibroblasts (4) in the airway wall. A proportion of asthmatic patients suffer from severe or

72 refractory asthma which is difficult to control despite receiving high doses of inhaled and

73 sometimes oral corticosteroids (5). These patients show increased airway wall remodeling,

74 with augmented subepithelial fibrosis and ASM thickening, which may contribute to the

75 persistent airflow obstruction in these patients (6-8).

76 Fibrocytes are bone marrow-derived circulating mesenchymal progenitor cells that express

77 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 response

79 to inflammation, and home on to the airway wall where they differentiate into

80 myofibroblasts. One mediator of fibrocyte migration to the lungs is the C-C chemokine

81 receptor 7 (CCR7) (10;11). CCR7 is expressed on circulating fibrocytes (12), while the expression

82 of its ligand, chemokine (C-C) motif ligand (CCL)-19, is increased in the ASM of patients

83 with asthma which implicates an important role of CCR7 in the homing of fibrocytes to

84 asthmatic airways (13). Fibrocytes are more abundant in the circulation of asthma subjects

85 with chronic airflow obstruction, and they have been localised to the ASM compartment in

86 the airway wall of patients with severe refractory asthma (12;14). At the tissue site, fibrocytes

87 can differentiate into myofibroblasts (15) which mediate subepithelial fibrosis through the

88 release of extracellular matrix (ECM) proteins (4) and may also contribute to increased ASM

89 mass (16).

90 Corticosteroid insensitivity is a feature of severe asthma. Corticosteroids are less effective in

91 suppressing the release of pro-inflammatory cytokines from peripheral blood mononuclear

92 cells (PBMCs) (17;18) and alveolar macrophages (19) from patients with severe asthma. ASMCs Lo 5

93 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 with

95 severe refractory asthma, and PBMCs exposed to interleukin (IL)-17 and IL-23 are resistant

96 to the induction of cell cycle arrest and apoptosis by corticosteroids (21-23). However, the effect

97 of corticosteroids on fibrocytes from patients with severe asthma is not known.

98 We hypothesized that patients with severe asthma have higher numbers of circulating

99 fibrocytes with increased differentiation potential and decreased sensitivity to the effects of

100 corticosteroids. We therefore determined the number of total and differentiating fibrocytes in

101 the non-adherent non-T (NANT) cell fraction of PBMCs of healthy subjects and patients with

102 non-severe and severe asthma, immediately after isolation and also following culture. We

103 also compared the expression of the glucocorticoid receptor (GR) in fibrocytes and the effect

104 of dexamethasone on the number of fibrocytes and the expression of CCR7 in these cells. The

105 effect of dexamethasone on apoptosis was also studied in fibrocytes isolated from the

106 adherent fraction of PBMCs.

112 MATERIALS AND METHODS

113 Study population

114 Healthy subjects and patients with non-severe and severe asthma were recruited. Subjects Lo 6

115 with FEV1 reversibility of less than 12% or with a provocative concentration of methacholine

116 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 the

118 presence of at least one of two major criteria for corticosteroid usage, and at least two minor

119 criteria (24). Patients with non-severe asthma had perfect control of their symptoms using

120 inhaled beclomethasone (0-1000 µg/d or equivalent). Current smokers and ex-smokers with

121 a smoking history of greater than 5 pack-years were excluded. All patients provided informed

122 consent and the study has been approved by the National Research Ethics Service Committee

123 London - Chelsea (REC 08/H0708/109).

124 Isolation of circulating fibrocytes from the non-adherent non-T (NANT) cell fraction

125 Non-adherent non-T (NANT) cells were isolated from peripheral blood as previously

126 described (12). PBMCs were isolated from whole blood using density gradient centrifugation

127 with Ficoll-PaqueTM PLUS (GE HealthCare, Uppsala, Sweden). Adherent cells were removed

128 by adherence and the non-adherent cell fraction was depleted of T cells using magnetic beads

129 coated with anti-CD3 mAb (Miltenyi Biotec, Auburn, California). The resulting NANT cells

130 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 the

132 presence or absence of the required treatments. The number of viable NANT cells was

133 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 cells

136 were fixed using 0.5% paraformaldehyde, washed twice with PBS and permeabilised using

137 BD FACS™ Permeabilizing Solution 2 (BD Biosciences, San Jose, California). Cell pellets

138 were incubated with mouse anti-human collagen I antibody (Millipore Corporation, Lo 7

139 Temecula, California) followed by a fluorescein isothiocyanate (FITC)-conjugated anti-

140 mouse secondary antibody (DAKO A/S, Glostrup, Denmark). Cell pellets were then

141 incubated with an allophycocyanin (APC)-conjugated mouse-anti-human CD45 antibody

142 (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 analysed

144 using a BD FACSCantoTM II flow cytometer. The relevant isotype antibody controls were

145 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 and

150 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 described

152 above.

153 Determination of GR expression in fibrocytes

154 NANT cells were fixed, stained with an APC-conjugated mouse anti-human CD45 antibody

155 (BD Biosciences) and permeabilised as described above. Cell pellets were then incubated

156 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-rabbit

158 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 staining

161 Apoptosis of NANT cells was analysed by Annexin V and propidium iodide (PI) staining Lo 8

162 using the FITC Annexin V/Dead cell apoptosis kit (Invitrogen) according to the

163 manufacturer’s instructions. Briefly, NANT cells were treated as required, washed in PBS

164 and then were incubated with FITC-conjugated annexin V and PI, and analysed by flow

165 cytometry. Annexin V-/PI- were considered live cells, Annexin V+/PI- as apoptotic and

166 annexin V+/PI+ as late apoptotic cells.

167 Statistical analysis

168 Data are presented as mean ± standard error of the mean (SEM). Statistical analysis was

169 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 severe

172 asthmatic groups, followed by Dunns post test. Correlations between parameters were

173 determined by Spearman’s rank correlation. p<0.05 was considered as statistically

174 significant.

176 RESULTS

177 Increased circulating fibrocytes in patients with severe asthma

178 Immediately after isolation there was no significant difference in the number of NANT cells

179 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 of

181 fibrocytes compared to that of non-severe asthma patients and healthy subjects, whilst the

182 percentage of circulating fibrocytes in non-severe asthma patients was higher than in healthy

183 subjects (Figure 1A and B). These differences were thus reflected in the absolute number of

184 fibrocytes per mL of blood which was increased in severe asthmatics compared to non-severe

185 asthmatics and healthy subjects, but also in non-severe asthmatics compared to healthy Lo 9

186 subjects (Figure 1C). The number of circulating fibrocytes in severe asthmatics who were on

187 oral prednisolone was not significantly different from that of patients who were not (See

188 Figure E2 in the Online Repository), suggesting that systemic corticosteroid therapy did not

189 affect the number of circulating fibrocytes. Moreover, the number of fibrocytes per mL of

190 blood showed an inverse correlation with the pre-bronchodilator FEV1% (Figure 1D) and

191 FEV1/FVC ratio (Figure 1E).

192 Increased differentiation of fibrocytes from patients with severe asthma

193 We observed an increase in the number of fibrocytes in the NANT cells from healthy subjects

194 and patients with non-severe asthma following culture, peaking after 3 days in culture, whilst

195 the number of fibrocytes from patients with severe asthma did not increase (Figure 2A). We

196 employed single α-SMA staining to identify differentiating fibrocytes as we have established

197 that the majority cells in the NANT cell population expressing α-SMA are fibrocytes (data

198 not shown). The number of differentiating fibrocytes in the NANT cells of healthy subjects

199 increased with time, peaking after 3 days (Figure 2B). The changes in α-SMA-positive cells

200 were followed by an increase in adherent “spindle-shaped” cells, which peaked after 10 days

201 in culture, indicating that fibrocytes can fully differentiate into myofibroblasts-like cells (See

202 Figure E3 in the Online Repository). We observed an increase in the number of

203 differentiating fibrocytes in the NANT cells of all groups, which also peaked after 3 days in

204 culture (Figure 2B). However, the number of differentiating fibrocytes in the NANT cells

205 from patients with severe asthma was higher compared to healthy subjects and patients with

206 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 reduction

209 in the number of fibrocytes and differentiating fibrocytes from healthy subjects and patients Lo 10

210 with non-severe asthma, but had no effect on those from patients with severe asthma (Figures

211 3A and 3B). Dexamethasone (10-8-10-6 M) also reduced the number of the total NANT cells

212 from healthy subjects and patients with non-severe asthma but not those from patients with

213 severe asthma (Figure 3C).

214 We determined the percentage of apoptotic NANT cells both at baseline and after

215 dexamethasone treatment. Immediately after isolation, the percentage of live and apoptotic

216 cells was similar in all groups (Figure 3D). After 3 days in culture, untreated NANT cells

217 from patients with severe asthma contained a higher percentage of live cells and a lower

218 percentage of early apoptotic cells compared to healthy subjects, suggesting increased

219 survival of severe asthmatic NANT cells (Figures 3E). Dexamethasone caused a reduction in

220 the percentage of live cells whilst increasing the percentage of early and late apoptotic cells

221 in NANT cells from healthy subjects (Figure 3F) and patients with non-severe asthma (Figure

222 3G). However, dexamethasone did not modulate the percentage of live and apoptotic NANT

223 cells from patients with severe asthma (Figure 3H). Similarly, when the data was expressed

224 as a total percentage of apoptotic cells, NANT cells from patients with severe asthma showed

225 a higher percentage of live, and a lower percentage of apoptotic, cells compared to those from

226 healthy subjects (See Table E1 in the Online Repository). Moreover, dexamethasone caused a

227 significant decrease in the percentage of total apoptotic cells from healthy subjects and

228 patients with non-severe asthma but had no effect on those from patients with severe asthma

229 (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 antagonist

232 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 11

234 No significant difference was observed in the baseline percentage of CCR7-positive

235 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 cells

238 from patients with non-severe asthma, but had no significant effect on those from patients

239 with severe asthma. Thus, the expression of CCR7 on the fibrocytes of patients with severe

240 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 healthy

243 subjects and patients with non-severe and severe asthma (Figure 6A). Both the percentage of

244 GR-positive fibrocytes (Figure 6B) and the GR MFI ratio (Figure 6C) were lower in patients

245 with severe asthma compared to healthy subjects and patients with non-severe asthma. The

246 percentage of GR-positive cells and GR MFI ratio were also reduced in the whole NANT cell

247 isolated from patients with severe asthma compared to healthy subjects and patients with

248 non-severe asthma (See Figure E4 in the Online Repository).

251 Effect of dexamethasone on fibrocytes isolated from adherent PBMCs

252 The percentage of fibrocytes and differentiating fibrocytes in adherent PBMCs increased in a

253 time-dependent manner peaking after 10 days in culture (See Figure E5 in the Online

254 Repository). Treatment with dexamethasone (10-7 M) for 72 hrs led to a decrease in the

255 number of fibrocytes and differentiating fibrocytes from healthy subjects, and this effect was

256 prevented by RU486 (10-6 M) (Figures 7A and 7B). Dexamethasone (10-7 M) also reduced the

257 number of fibrocytes and differentiating fibrocytes from patients with non-severe asthma but

258 had no significant effect on those from patients with severe asthma (Figures 7C and 7D). Lo 12

259 Thus, fibrocytes isolated from the adherent fraction of PBMCs of severe asthmatic patients

260 are also resistant to corticosteroid-induced apoptosis.

272 DISCUSSION

273 We have demonstrated that, immediately after isolation, there is a higher number of

274 fibrocytes in the circulating blood of patients with severe asthma compared to healthy

275 subjects and patients with non-severe asthma. Furthermore, we showed that, when placed in

276 culture, fibrocytes from patients with severe asthma have a greater capacity to differentiate

277 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 13

280 patients with severe asthma. In line with these findings, dexamethasone reduced the

281 percentage of live NANT cells, and increased the percentage of apoptotic NANT cells from

282 healthy subjects and patients with non-severe asthma but had no effect on those from patients

283 with severe asthma. Fibrocytes from patients with severe asthma were also resistant to the

284 inhibition of CCR7 expression by dexamethasone and showed reduced GR expression

285 compared to those from healthy subjects and patients with non-severe asthma. Thus, patients

286 with severe asthma have a higher number of circulating fibrocytes that have a greater

287 capacity to differentiate into myofibroblasts while showing relative corticosteroid

288 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 more

291 abundant in the circulation of patients with severe asthma and have an increased capacity to

292 differentiate into myofibroblasts compared to healthy subjects and patients with non-severe

293 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 and

295 in the ASM bundles of patients with asthma of all severities, suggesting that fibrocytes can

296 migrate to the airway wall of these patients and differentiate into myofibroblasts (14).

297 Myofibroblasts exhibit a phenotype between fibroblasts and smooth muscle cells that

298 involves secretion of ECM proteins (25) but also expression of smooth muscle-specific

299 proteins (26). The number of myofibroblasts in the airways of patients with asthma was found

300 to be correlated with the extent of subepithelial fibrosis (4). Myofibroblasts have also been

301 detected in the ASM bundles of patients with asthma, therefore possibly contributing to the

302 increased ASM mass (16). Thus, patients with severe asthma may have a greater pool of

303 circulating fibrocytes which can readily differentiate into myofibroblasts upon migration to

304 the airway wall, contributing to the remodelling process. Lo 14

305 Patients with severe asthma show exaggerated airway remodelling despite being on high

306 doses of inhaled and sometimes oral corticosteroid treatment (6-8). We, therefore, determined

307 the effect of corticosteroids on the function of fibrocytes isolated from these patients. Our

308 data show a reduction in the number of fibrocytes and differentiating fibrocytes from healthy

309 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 the

311 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 reported

313 dexamethasone-induced inhibition of the proliferation of circulating fibrocytes of healthy

314 subjects (27). Dexamethasone also led to a reduction in the expression of CCR7 in the

315 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 CCR7

318 in fibrocytes from patients with severe asthma indicating that these cells show relative

319 corticosteroid insensitivity. This may result from the lower GR expression levels we observed

320 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 mechanisms

323 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 to

325 induce apoptosis and reduce the number of total NANT cells from patients with severe

326 asthma. NANT cells from patients with severe asthma also exhibited lower levels of GR

327 expression compared to healthy subjects and patients with severe asthma. Thus, the relative

328 corticosteroid insensitivity we observed in fibrocytes from patients with severe asthma also

329 extends to non-fibrocyte cells. Nonetheless, as we have shown that NANT cells from patients Lo 15

330 with severe asthma show increased survival, we cannot exclude the possibility that the failure

331 of dexamethasone to reduce the number these cells is a result of an inherent resistance to

332 apoptosis.

333 We have previously demonstrated relative corticosteroid insensitivity in PBMCs (17), alveolar

334 macrophages (19) as well as ASMCs (20) from patients with severe asthma. We now extend this

335 observation to show that in patients with severe asthma, circulating progenitor cells such as

336 fibrocytes may also exhibit an altered response to corticosteroids. This finding may have

337 important implications in understanding the mechanisms underlying the increased

338 remodelling observed in these patients.

339 The most commonly described method of isolating fibrocytes from blood uses the adherent

340 fraction of PBMCs (31;32). However, fibrocytes are also abundant in the non-adherent fraction

341 of PBMCs (12;33). We found that fibrocytes isolated from the adherent fraction of PBMCs of

342 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 pattern

344 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 study

347 involving tracking fluorescence-labeled fibrocytes in a mouse model of allergic asthma has

348 shown recruitment of fibrocytes from the circulation to the bronchial tissue and

349 differentiation into myofibroblasts in response to allergen exposure (15). Similar studies in

350 animal models of severe asthma may provide a clearer picture of the involvement of

351 fibrocytes in the development of airway remodelling in this disease.

352 Fibrocytes are potentially important cells involved in the airway wall remodeling in asthma

353 (10). Our study demonstrates that patients with severe asthma have elevated numbers of Lo 16

354 circulating fibrocytes which show an aberrant phenotype involving a heightened ability to

355 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 airway

357 remodelling in severe asthma and could be an important therapeutic target.

358 359 ACKNOWLEDGEMENTS 360

361 We thank João Pedro Carvalho da Purificacao Rocha for recruiting the subjects and

362 Kirandeep K. Chana, Po-Jui Chang, Chih-Ming Weng, and Kang-Yun Lee for their technical

363 help and advice.

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479 FIGURE LEGENDS

480 Figure 1. Number of circulating fibrocytes in freshly isolated NANT cells from healthy

481 subjects and patients with non-severe and severe asthma. Representative flow cytometry

482 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) was

485 determined. Horizontal lines represent the median for each group. (D-E) Correlation of

486 fibrocyte numbers with FEV1 % predicted (D) and FEV1/FVC ratio (E) was determined using

487 Spearman’s rank correlation. * p<0.05, ** p<0.01, *** p<0.001. RS: Spearman’s rank

488 correlation.

489 Lo 20

490 Figure 2: Number of fibrocytes and differentiating fibrocytes in cultured NANT cells

491 from healthy subjects and patients with non-severe and severe asthma. The number of

492 fibrocytes (Col I+/CD45+ cells; A) and differentiated fibrocytes (α-SMA+ cells; B) were

493 determined in NANT cells from healthy subjects (n=5-18) and patients with non-severe

494 (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 healthy

496 day 3. $$ p < 0.01 vs non-severe asthma day 3.

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) and

500 severe asthma (n=12) were cultured for 3 days in the presence or absence of dexamethasone

501 (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 change

503 compared to untreated control. (D-E) The percentage of live, and early and late apoptotic

504 NANT cells from healthy subjects (H; n=6) and patients with non-severe (NSA; n=6) and

505 severe asthma (SA; n=6) was determined immediately after isolation (D), or after 3 days in

506 culture in the absence (E) or presence of dexamethasone (10-7M; F-H). Data points and bars

507 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.

510 Figure 4: Effect of GR inhibition on dexamethasone-induced reduction in fibrocyte

511 number. NANT cells from healthy subjects were treated with dexamethasone (10-7M) in the

512 presence or absence of RU486 (10-6-10-5 M) and total NANT cell (A), fibrocyte (Col

513 I+/CD45+ cells; B) and differentiating fibrocyte (α-SMA+ cells; C) numbers were Lo 21

514 determined after 3 days. Bars represent mean ± SEM of 6 donors. * p<0.05.

516 Figure 5: Effect of dexamethasone on CCR7 expression in fibrocytes from patients with

517 non-severe and severe asthma. (A) Representative flow cytometry scatter plots from one

518 experiment per group are shown. (B) The percentage of CCR7+ fibrocytes (Col

519 I+/CD45+/CCR7+ cells) was determined in NANT cells from patients with non-severe

520 (NSA; n=10) and severe asthma (SA; n=12) immediately after isolation or after 3 days in

521 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 fluorescence

523 intensity (MFI) ratio (D) after treatment with dexamethasone (10-7M) for 3 days was

524 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 one

528 experiment per group are shown. The percentage (B) and median fluorescence intensity

529 (MFI) ratios (C) of GR+ fibrocytes (Col I+/CD45+/GR+ cells) were determined in freshly

530 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.

533 Figure 7. Effect of dexamethasone on the number of fibrocytes derived from adherent

534 PBMCs. (A-B) Adherent PBMCs from healthy subjects (n=6) were placed in culture for 3

535 days followed by incubation with dexamethasone (10-7 M), in the presence or absence of

536 RU486 (10-6 M), for a further 3 days. The number of fibrocytes (Col I+/CD45+ cells; A) and

537 differentiating fibrocytes (α-SMA+ cells; B) was determined. (C-D) Adherent PBMCs from Lo 22

538 healthy subjects (n=6), and patients with non-severe (n=5) and severe asthma (n=6) were

539 placed in culture for 3 days followed by incubation with dexamethasone (10-7 M) for a further

540 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.