CAPSULE SUMMARY: Out of all type-2 immune cells, only ILC2s withstand corticosteroid treatment due to a reduced pro-apoptotic machinery. The findings may advance the understanding on the mechanism in steroid-resistant diseases and facilitate the development of improved therapies

Chen, Y-L

Go, S

Hardman, C

Klenerman, P

Liu, W

Luo, J

Nahler, J

Ogg, G

Pavord, Ian

Reed, L

Shrimanker, R

Stöger, L

Tolkovsky, AM

Wu, Y

Xue, Luzheng

Ye, Y

Zeng, N

Journal of Allergy and Clinical Immunology

English

Pavord, ID

Xue, L

Oxford University Research Archive (ORA)

Resistance to apoptosis underpins corticosteroid-insensitivity of group 2 innate lymphoid cells

Oxford University Research Archive

Luo et al. 1 Notice: No Marked Manuscript has been provided as this manuscript has been 1 reformatted from an Original Article to a Letter to the Editor. 2  3 TITLE PAGE 4  5 Resistance to apoptosis underpins corticosteroid-insensitivity of group 2 6 innate lymphoid cells 7  8 Jian Luo, MD, PhD,a,§ Yanqiu Wu, MSc,a,§ Rahul Shrimanker, MD,a,§ Simei Go, 9 MSc,a,§ Yuan Ye, MSc,a,§ Clare Hardman, PhD,b Janina Nahler, BSc,b Yi-Ling Chen, 10 DPhil,b Linda Stöger, MSc,a Ni Zeng, BM,a Wei Liu, MD, PhD,a Lara Reed,a Aviva M. 11 Tolkovsky, PhD,c Paul Klenerman, FMedSci,d Graham Ogg, DPhil, FRCP,b Ian D. 12 Pavord, FMedSci,a Luzheng Xue, PhD,a 13  14 aRespiratory Medicine Unit, and Oxford NIHR Biomedical Research Centre, 15 University of Oxford, UK 16 bMRC Human Immunology Unit, and Oxford NIHR Biomedical Research Centre, 17 University of Oxford, UK 18 cDepartment of Clinical Neurosciences, University of Cambridge, UK 19 dTranslational Gastroenterology Unit and Peter Medawar Building, University of 20 Oxford, UK. 21  22 § These authors contributed equally to this work. 23  24 Corresponding author 25 Luzheng Xue 26 Luo et al. 2 Respiratory Medicine Unit, Nuffield Department of Medicine, University of Oxford, 27 Headley Way, Oxford OX3 9DU, UK. 28 Telephone: (+44) 1865 221349 29 Email: luzheng.xue@ndm.ox.ac.uk 30  31 Declaration of all sources of funding: NIHR Oxford Biomedical Research Centre 32 Programme (PK, GO, IDP, LX and SG), Wellcome Trust (PK-WT109965MA), MRC 33 (GO). 34  35 Conflict of interest: The authors have declared that no conflict of interest exists. 36  37 Capsule Summary 38 Out of all type-2 immune cells, only ILC2s withstand corticosteroid treatment due to 39 a reduced pro-apoptotic machinery. The findings may advance the understanding 40 on the mechanism in steroid-resistant diseases and facilitate the development of 41 improved therapies. 42  43 Key Words 44 Asthma, corticosteroids, ILC2, Th2, Tc2, apoptosis, Bcl-2, mitochondria 45   46 Luo et al. 3  47 Abbreviations used 48 CRTH2: Chemoattractant receptor-homologous molecule expressed on Th2 cells 49 GR: glucocorticoid receptor 50 HSP: heat shock protein 51 ILC2: Group 2 innate lymphoid cells 52 MDR1: multi-drug resistance protein 1 53 MMP: mitochondrial membrane potential 54 MTG: MitoTracker Green 55 MTO: MitoTracker Orange 56 OCS: oral corticosteroids 57 Tc2: type-2 CD8+ cytotoxic T cells 58 Th2: type-2 CD4+ T helpers  59 Luo et al. 4 To the Editors: 60 Steroid-insensitivity is one of the major clinical problems in severe asthma, and a 61 better understanding of the mechanisms is important.1 Given that type-2 cells, 62 including type-2 CD4+ T helpers (Th2), type-2 CD8+ cytotoxic T cells (Tc2) and 63 group 2 innate lymphoid cells (ILC2s) are all contributors to the pathogenesis of 64 asthma,2,3 it is important to define both their relative resistance to corticosteroids 65 and the underpinning mechanisms.4 We characterised the effects of corticosteroids 66 on three major types of human type-2 cells (CD3+CD4+CRTH2+ Th2, 67 CD3+CD8+CRTH2+ Tc2 and Lin-CD45highCD127+CRTH2+ ILC2) in vivo and in vitro. 68 We further defined the potential mechanisms contributing to the insensitivity. 69 We first compared the blood counts of type-2 cells in patients with severe 70 eosinophilic asthma before and after oral corticosteroid (OCS) treatment with 71 prednisolone (Fig. 1A-D, Table E1). After a period (7-33 days) of OCS, the blood 72 eosinophil counts were markedly reduced (from 477.9±184.2 x106 to 66.4±87.9 x106 73 cells/L, p<0.0003), while the neutrophil counts increased. The Th2 and Tc2 cell 74 counts were decreased from 37.1±45.5 x106 to 12.6±18.6 x106 cells/L (p=0.02) and 75 from 45.9±79.8 x106 to 6.8±6.2 x106 cells/L (p=0.027), respectively (Fig. 1A,C). In 76 contrast, ILC2s were increased, from 0.4±0.43 x106 to 0.57±0.49 x106 cells/L 77 (p=0.019), representing an increase in the proportion of ILC2s from 1.4% to 8.3% of 78 total blood type-2 cells (Fig. 1D). 79  To determine if ILC2s’ tolerance to corticosteroids is cell-autonomous, we 80 tested the effect of corticosteroids on apoptosis induction in cultured type-2 cells 81 from healthy blood in vitro. Incubation with dexamethasone for 16 hours induced 82 apoptosis in all three cell types in a dose-dependent manner but the maximal ILC2 83 response to dexamethasone was significantly less than Th2/Tc2 cells (Fig. 1E). 84 Luo et al. 5  To understand whether ILC2s’ resistance to apoptosis induction is specific to 85 corticosteroids, cells were exposed to staurosporine (Fig. 1F,G). The ILC2 apoptotic 86 response to incubation with staurosporine for 4 hours was significantly less than 87 Th2/Tc2 cells (Fig. 1F). When cells were treated with staurosporine (10 µM) for 88 longer periods, ILC2s showed a significantly delayed response compared with 89 Th2/Tc2 cells (Fig. 1G). By 9 hours, similar ratios of apoptosis were detected in all 90 three cell types, suggesting that ILC2s are as capable of undergoing apoptosis as 91 the other type-2 cells, but their apoptotic efficiency is reduced. 92 Corticosteroid-insensitivity of ILC2s appeared to be restricted to apoptotic 93 pathways, as dexmethasone attenuated chemotaxis induced by prostaglandin D2 94 (Fig. E1A) or IL-13/5 production induced by prostaglandin D2 or PMA/Ionomycin 95 (Fig. E1B,C) similarly in all three cell types. 96  ILC2 resistance to corticosteroid-induced apoptosis was not due to a reduced 97 expression of the glucocorticoid receptor (GR), as no significant difference of GR 98 expression between the cell types was detected at either protein or mRNA (NR3C1) 99 levels in blood ex vivo or cultured cells (Fig. E2A-C). It has been suggested that 100 heat shock protein (HSP) 90 and 70 molecular chaperones are required for GR 101 function, and HSP70 is also an anti-apoptotic protein.5 However, no difference in 102 HSP90 expression was detected (Fig. E2A-C), whereas HSP70 expression in ILC2s 103 detected by flow cytometry was significantly lower than in Th2/Tc2 in fresh blood 104 (Fig. E2A right panel); however, HSP70 levels were equivalent in cultured cells (Fig. 105 2D,E and E2B,C right panels). Thus, it is unlikely that HSP70 contributes to 106 resistance of ILC2s to dexamethasone-induced apoptosis. Reduced expression of 107 multi-drug resistance protein 1 (MDR1) could potentially reduce the efficacy of 108 steroids by transporting drugs out of cells.6 However, no significant difference in 109 Luo et al. 6 MDR1 protein expression was detected between the three cell types (Fig. E2D). 110 Importantly, the MDR1 inhibitors, Tariquidar or Zosuquidar, failed to alter the 111 sensitivity of ILC2s to dexamethasone (Fig. E2E). 112 We next characterised the cell-intrinsic apoptotic pathways in type-2 cells.7 113 The level of anti-apoptotic protein Bcl-2, detected by flow cytometry in fresh blood, 114 was slightly higher in ILC2s compared to Th2 and Tc2 cells (Fig. 2A). Anti-apoptotic 115 Bcl-xL expression was not significantly different among the cell types. In contrast, 116 the relative expression of the major pro-apoptotic Bcl-2 proteins BAX and BAK were 117 significantly lower in ILC2s. In cultured cells, mRNA expression of BCL2 and 118 BCL2L1 (gene for Bcl-xL) were higher in ILC2s, but no differences were detected in 119 BAX1 and BAK1 (Fig. 2B). Comparison of Bcl-2 family expression within each cell 120 type showed that the ratios of pro-apoptotic to anti-apoptotic Bcl-2 mRNAs were 121 significantly lower in ILC2s (Fig. 2C, E3A). These differences were further confirmed 122 by proteome profiler array (Fig. 2D, E3B). In keeping with slightly higher expression 123 of Bcl-2 and less BAD/BAX, the expressions of cleaved (active)-caspase 3, HSP27 124 and HSP60 were also lower in ILC2s compared to Th2 cells (Fig. 2E). No 125 differences in the expression of inhibitor of apoptotic proteins (IAPs including cIAP-126 1, cIAP-2 and XIAP) were detected between the cell types. Similar to our qPCR 127 results, the ratios of BAD or BAX to Bcl-2 were significantly lower in ILC2s (Fig. 2F). 128 Dexamethasone (treated for 8 h) did not significantly change the ratios of pro-/anti-129 Bcl2 proteins in the cells (Fig. E3C-F), indicating that their expression is not directly 130 determined by GR signaling, but is rather an intrinsic property of these cells. 131  To test whether the altered ratios of pro-/anti-apoptotic Bcl2 proteins play a 132 functional role in apoptosis resistance, we employed Bcl-2 inhibitors.8 ABT263 or 133 ABT737, at 1 nM, which did not induce apoptosis alone (Fig. E4), was sufficient to 134 Luo et al. 7 sensitize ILC2s to dexamethasone-induced apoptosis (Fig. 2G) as well as to 135 staurosporine (Fig. 2H), confirming that the low anti-/pro-apoptotic Bcl-2 family 136 protein ratio determines the reduced response of ILC2s to dexamethasone. 137  BAX and BAK function by  permeabilizing the mitochondria to release 138 apoptotic proteins,7 and  mitochondrial content is strongly correlated with apoptosis 139 sensitivity.9 Interestingly, mitochondrial content measured with MitoTracker Green 140 (MTG,  a mitochondrial membrane potential (MMP)-independent dye, Fig. E5A) or 141 an antibody against the outer membrane protein TOMM20 showed lower 142 mitochondrial mass in ILC2s compared with Th2/Tc2 cells in both ex-vivo blood and 143 cultured cells (Fig. 2I,J), which was also consistent with the reduction of HSP60, a 144 mitochondrial chaperonin, in ILC2s (Fig. 2D,E). Low MTG staining was not due to 145 loss of MMP, verified using MitoTracker Orange (MTO, a MMP-dependent dye, Fig. 146 E5B). Therefore, fewer mitochondria may act in concert with lower pro-/anti-147 apoptotic Bcl-2 protein ratios to cause corticosteroid-resistance in ILC2s. 148 To understand whether corticosteroid-resistance and the reduced propensity 149 to apoptosis are unique features of ILC2s, we also investigated other related cells 150 (Fig. E6). As expected, T lymphocyte counts in blood samples were significantly 151 reduced after OCS (Fig. E6A); however, similar to ILC2s, other ILCs were not (Fig. 152 E6B). The expression profiles of Bcl-2 proteins (Fig. E6C,D) and the mitochondrial 153 content (Fig. E6E,F,G) in CD3/4/8 cells were similar to those of Th2/Tc2 cells. 154 However, CRTH2-ILCs (ILC1/3s) were similar to ILC2s in that pro-apoptotic BAX 155 and BAK to Bcl-2 ratios, and mitochondrial mass were lower compared to T cells. 156 Collectively, our data show that ILC2 survival is corticosteroid-insensitive 157 both in vivo and in vitro, which is associated with intrinsically reduced pro-apoptotic 158 machinery including pro-apoptotic Bcl-2 proteins and mitochondria. Our 159 Luo et al. 8 observations provide a better understanding of the molecular mechanisms 160 mediating corticosteroid-resistance in ILC2s. We also revealed for the first time the 161 corticosteroid-tolerance features of other ILCs. These new findings are of potential 162 clinical value and provide further insight into treatment of ILC2 (and ILC)-mediated 163 inflammatory diseases. 164 165 Luo et al. 9 REFERENCES 166 1. 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Glucocorticoid 179 receptor function regulated by coordinated action of the Hsp90 and Hsp70 180 chaperone cycles. Cell 2014;157:1685-97. 181 6. Gruol DJ, Bourgeois S. Expression of the mdr1 P-glycoprotein gene: a 182 mechanism of escape from glucocorticoid-induced apoptosis. Biochem Cell Biol 183 1994;72:561-71. 184 7. Singh R, Letai A, Sarosiek K. Regulation of apoptosis in health and disease: the 185 balancing act of BCL-2 family proteins. Nat Rev Mol Cell Biol 2019;20:175-93. 186 8. Tian B, Xia L, Bao Z, Zhang H, Xu Z, Mao Y, et al. Bcl-2 inhibitors reduce steroid-187 insensitive airway inflammation. J Allergy Clin Immunol 2017;140:418-30. 188 Luo et al. 10 9. Márquez-Jurado S, Díaz-Colunga J, das Neves RP, Martinez-Lorente A, Almazán 189 F, Guantes R, et al. Mitochondrial levels determine variability in cell death by 190 modulating apoptotic gene expression. Nat Commun 2018;9:389.  191 Luo et al. 11 Figure Legends 192  193 FIG 1. ILC2s were less sensitive than Th2 and Tc2 cells to corticosteroids in clinical 194 treatment and in vitro apoptosis assay. (A) A representative flow cytometric gating 195 example to compare the frequencies of Th2 (CD3+CD4+CRTH2+), Tc2 196 (CD3+CD8+CRTH2+) and ILC2 (Lin-CD45highCD127+CRTH2+) cells in the blood from 197 a patient with severe asthma before and after oral prednisolone treatment. 198 Eosinophils (EOS) and Neutrophils (NEU) were used as control. (B,C) Comparison 199 Luo et al. 12 of blood counts of (B) eosinophils and neutrophils, (C) Th2, Tc2 and ILC2s before 200 and after prednisolone-treatment. (D) Average proportions of Th2, Tc2 and ILC2 in 201 total type-2 cells before and after prednisolone-treatment. Paired t tests with two 202 tailed. (E-G) Annexin-V+ apoptotic cells in cultured Th2, Tc2 and ILC2 cells in 203 response to (E) 16 h-treatment of various concentrations (left panel) or 1 µM (right 204 panel) of dexamethasone, or (F) 4 h-treatment of various concentrations (left panel) 205 or 30 µM (right panel) of staurosporine, or (G) different length of time incubation with 206 10 µM staurosporine detected with flow cytometry. **** p<0.0001, *** p<0.001, 207 **p<0.01, * p<0.05; n=10-12 for D and E, n=4 for F, n=3 for G. 208  209 Luo et al. 13  210 FIG 2. ILC2s had reduced pro-apoptotic machinery including pro-apoptotic Bcl-2 211 proteins and mitochondria compared with Th2 and Tc2 cells. (A) Levels of Bcl-2 212 family proteins including Bcl-2, Bcl-xL, BAX and BAK in blood type-2 cells compared 213 with flow cytometry. (B) mRNA levels of Bcl-2 family members in cultured type-2 214 Luo et al. 14 cells compared with qPCR. (C) Comparison of ratios of pro- to anti-apoptotic Bcl-2 215 family genes in cultured type-2 cells. (D) Expression of apoptosis-related proteins 216 including BAD, BAX, Bcl-2, Bcl-xL, pro-caspase 3 (Casp3), cleaved-Casp3, 217 HSP27/60/70, cIAP-1/-2 and XIAP in cultured Th2 and ILC2s detected with 218 Proteome profiler array. (E) Quantitative comparison of these proteins. (F) 219 Comparison of ratios of pro- against anti-apoptotic Bcl-2 proteins in Proteome 220 Profiler Array. (G,H) Enhancement of ILC2 apoptosis in response to 16 h-treatment 221 of various concentrations (G left panel) or 1 µM (G right panel) dexamethasone, or 3 222 h-treatment of 10 µM staurosporine (H) by Bcl-2 inhibitors, ABT263 and ABT737. 223 (I,J) MTG, MTO or anti-TOMM20 staining for flow cytometry in blood type-2 cells (I) 224 or cultured type-2 cells (J). The three types of staining were compared by converting 225 the data to index of the staining levels (I,J right panels). ****p<0.0001, *** p<0.001, 226 ** p<0.01, * p<0.05; n=6-13 for C, n=3 for D-F, n=7 for G, n=5-18 for I and J. 227 

Resistance to apoptosis underpins the corticosteroid insensitivity of group 2 innate lymphoid cells

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Resistance to apoptosis underpins the corticosteroid insensitivity of group 2 innate lymphoid cells

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