IL-33/ST2 axis promotes mast cell survival via BCLXL Jun-Xia W a n g 3'*1, Shinjiro Kaieda3b'1, Sarah A m eri3, Nadia Fishgal3, Daniel D w y e r3, A n th o n y Dellinger", C hristopher L. Kepley", M ichael F. Gurish3, and Peter A. Nigrovic a d ' 2 D ivision o f R h e u m a to lo g y, Im m u n o lo g y, an d A lle rg y , D e p a rtm e n t o f M e dicine, B righa m an d W o m e n 's H ospital, H arvard M e dical School B oston M A 02115 D e p a rtm e n t o f M e dicine, K u ru m e U n ive rsity School o f M e dicine, K u ru m e 830-0011, Japan; 'D e p a rtm e n t o f Nanoscience, J o in t School o f Nanoscience and N,a!?°te c h n o lo g y ' U n ive rsity N o rth C arolina a t G reensboro, G reensboro, NC 27412; an d dD ivision o f Im m u n o lo g y , D e p a rtm e n t o f M e d ic in e Boston C h ild re n 's H ospita l, H arva rd M e dical School, Boston, M A 02115 E dited by Cem Gabay, U n ive rsity H ospita l Geneva, Geneva, S w itze rla n d , and accepted by th e E d ito ria l Board June 11, 2014 (received fo r re v ie w M a rch 4, 2014) M a s t cells (M C ) a r e p o t e n t in n a te im m u n e cells t h a t a c c u m u la te in
derived MC (BMMC) (20-23). IL-10 can either promote or im pair survival depending on the type of MC studied (21, 22). r e c e p to r -r e la te d p r o te in ST2 a t h ig h le v e l, a n d th is IL-1 f a m ily Recently, substantial attention has focused on the role of c y to k in e b o th a c tiv a te s M C d ir e c tly a n d p rim e s t h e m t o re s p o n d to IL-33 in MC biology. A member of the IL-1 family, IL-33 is o t h e r p r o in f la m m a t o r y s ig n a ls . W h e t h e r IL -3 3 a n d ST2 p la y a ro le expressed primarily by stromal cells, such as epithelial cells, in M C s u rv iv a l re m a in s t o b e d e fin e d . In s k in -d e r iv e d h u m a n M C , endothelial cells, and fibroblasts, as well as by certain hema w e f o u n d t h a t IL -3 3 a t t e n u a t e d M C a p o p to s is w it h o u t a lte r in g topoietic cells, including MC themselves (24-26). IL-33 is an p ro life r a tio n , a n e ffe c t m e d ia te d p rin c ip a lly th r o u g h th e a n tia p o p “alarmin’’ released upon cell necrosis and also can be secreted t o t ic m o le c u le B -c e ll ly m p h o m a - X la r g e (B C L X L ). M u r i n e M C by live cells after mechanical stretching and via other incomplete d e m o n s t r a t e d a s im ila r m e c h a n is m , d e p e n d e n t e n t i r e l y o n ST2. ly defined mechanisms (27, 28). Its receptor ST2 (also called In lin e w it h th e s e o b s e rv a tio n s , S t2 ~ '~ m ic e e x h ib ite d re d u c e d IL-1 receptor-related protein 1) is highly expressed by MC. n u m b e rs o f tis s u e M C in in fla m e d a r th r itic jo in ts , in h e lm in th Acting via ST2, IL-33 participates in MC activation in murine in fe c te d in te s tin e , a n d in n o r m a l p e r ito n e u m . T o c o n fir m a n M C models of anaphylaxis and arthritis, serving both as a direct MC in trin s ic ro le f o r ST2 in v iv o , w e p e r fo r m e d p e r ito n e a l tr a n s f e r o f activator and as a factor that primes MC for enhanced cytokine W T a n d S t2 ~ '~ M C . In S t2 ~ '~ h o s ts t r e a t e d w it h IL -3 3 a n d in W T release upon subsequent stimulation by IgE and IgG (29-32). h o s ts s u b je c te d t o th io g ly c o lla te p e r ito n itis , W T M C d is p la y e d IL-33 also prominently modulates the MC phenotype, pro a c le a r s u r v iv a l a d v a n t a g e o v e r c o e n g r a f t e d S f 2 _ /~ M C . IL -3 3 moting accumulation of MC protease 6 (the murine ortholog of b lo c k a d e s p e c ific a lly a t t e n u a t e d th is s u rv iv a l a d v a n ta g e , c o n fir m human tryptase (3) in vitro and in vivo (33). in g IL -3 3 as t h e r e le v a n t ST2 lig a n d m e d ia tin g M C s u rv iv a l in v iv o . In cardiomyocytes and hepatocytes, IL-33 protects against T o g e t h e r , t h e s e d a t a r e v e a l a c e ll-in tr in s ic r o le f o r t h e IL -3 3 /S T 2 apoptosis (34, 35), raising the possibility of an analogous effect in a x is in t h e r e g u la t io n o f a p o p to s is in M C , i d e n t i f y i n g t h e r e b y MC. Improved in vitro survival has been observed in human a p r e v io u s ly u n a p p r e c ia t e d p a t h w a y s u p p o r t in g e x p a n s io n o f umbilical cord blood-derived MC exposed to IL-33 and in cul t h e M C p o p u la t io n w i t h i n f la m m a t io n . tured murine MC under certain conditions, but this effect is incompletely characterized, and its relevance in vivo remains a r th ritis | h e lm in th in fe c tio n unknown (36, 37). In the present study, we show that IL-33 promotes the survival of both human and murine MC, mediated ast cells (MC) are tissue-resident effector cells that con principally by up-regulation of the antiapoptotic factor BCLXL. tribute both to innate and adaptive immunity (1). MC help In vivo, IL-33 and ST2 play a nonredundant role in promoting defend against bacterial and helminthic infection (2, 3) and play a role in tissue remodeling (4, 5). MC can also contribute to Significance a variety of allergic and nonallergic inflammatory diseases, such as anaphylaxis, atopic dermatitis, asthma, inflammatory arthritis, M a s t cells (M C ) a r e lo n g -liv e d cells t h a t a c c u m u la te in in fla m e d psoriasis, and multiple sclerosis (6, 7). Under many of these tis s u e s . IL -3 3 h a s b e e n c h a r a c te riz e d e x te n s iv e ly as a M C a c ti conditions, the number of MC in affected tissues increases by v a t o r , b u t a c o n t r ib u t io n o f IL -3 3 a n d its r e c e p to r IL-1 re c e p to r tenfold or more, amplifying the contribution of MC-derived r e la t e d p r o te in S T2 t o M C s u r v iv a l r e m a in s u n a p p r e c ia t e d . mediators to ongoing inflammation (6-8). Regulation of the MC H e r e , w e s h o w t h a t IL -3 3 a t t e n u a t e s a p o p to s is o f h u m a n a n d population is poorly understood, and therapeutic intervention to m u r in e M C , p r in c ip a lly v ia t h e a n t ia p o p t o t ic m o le c u le B-cell limit MC accumulation potentially could attenuate injury asso ly m p h o m a -X la rg e (B C LX L). In v iv o , IL -3 3 a n d ST2 ca n c o n fe r ciated with inflammatory diseases (9, 10). a c e ll-in trin s ic s u rv iv a l a d v a n ta g e t o m u r in e M C , p a r tic u la r ly in The survival of mature MC in tissues depends on signals from t h e c o n t e x t o f in f la m m a tio n . T h e s e re s u lts id e n t if y t h e IL -3 3 / neighboring cells (11, 12). The single most important mediator in ST2 a x is as a n im p o r t a n t p a t h w a y s u p p o r tin g M C p e rs is te n c e this process is stem cell factor (SCF), a master regulator of MC in tis s u e s , ra is in g t h e p o s s ib ility t h a t th e r a p e u t ic t a r g e t in g proliferation, differentiation, survival, and activation. Mice with o f IL -3 3 c o u ld lim it t h e c o n t r ib u t io n o f M C t o c h ro n ic in genetic mutations affecting SCF or its receptor Kit have few or f la m m a t o r y d is ea ses . no MC in healthy or inflamed tissues, whereas activating muta tions of Kit give rise to pathologic mastocytosis (13-15). IL-3 is A u th o r contributions: J.-X.W., S.K., C.L.K., M.F.G., and P.A.N. designed research; J.-X.W., S.K., S.A., N.F., D.D., A.D., and M.F.G. perform ed research; A.D. and C.L.K. contributed another well-recognized MC growth factor (16). SCF/Kit and new reagents/analytic tools; J.-X.W., S.K., M.F.G., and P.A.N. analyzed data; and J.-X.W., IL-3 interface with several antiapoptotic pathways, up-regulating S.K., and P.A.N. w rote th e paper. the antiapoptotic protein B-cell lymphoma-2 (BCL-2) and down The authors declare no conflict o f interest. regulating the proapoptotic protein Bim (14, 17-19). Aside from This article is a PNAS Direct Submission. C.G. is a guest e d ito r invited by th e Editorial SCF/Kit and IL-3, other cytokines have been reported to support Board. MC survival, but their effects appear to be restricted to specific 1J.-X.W. and S.K. contributed equally to this w ork. MC subtypes. IL-4 promotes survival of intestinal MC via both 2To w hom correspondence should be addressed. E-mail: pnigrovic@ partners.org. BCL-2 and B-cell Iymphoma-X large (BCLXL) but may suppress This article contains supporting inform a tio n online a t www .pnas.org/lookup/suppl/doi: 10. survival in human cord blood MC as well as murine bone marrow1073/pnas. 1404182111/-/DCSupplemental. c h ro n ic a lly in fla m e d tissu e s. M C e x p re s s t h e IL -3 3 r e c e p to r IL-1
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than that of siRNA #1 (~60%) (Fig. SL4). Under SCF culture conditions, potent knockdown of either gene increased apoptosis by 1.4- to 1.7-fold (Fig. SIB) (16). The protective effect of IL-33 was almost abolished by silencing BCLXL, whereas BCL-2 knock down had a minor impact (Fig. ID). Taken together, these results indicate that IL-33 protects human MC from apoptosis primarily through expression of BCLXL. T h e IL-33 R e c e p to r ST2 Is R e q u ire d f o r th e P ro s u rv iv a l E ffe c t o f IL-33.
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g e n e e x p re s s io n . (A ) H u m a n s k in -d e riv e d M C a t 1 x 105/m L
w e r e tr e a t e d w i t h o r w i t h o u t IL-33 (1 0 n g /m L ) in t h e p re s e n c e o r a b s e n c e o f SCF f o r 3 d . M C a p o p to s is w a s assessed b y f l o w c y to m e tr y . (B) C ell d iv is io n w a s m e a s u re d b y CFSE d ilu t io n assay in liv e M C (A n n e x in V “ P r ) w i t h o r w i t h o u t IL-33 in t h e a b s e n c e o f SCF f o r 3 d . G ra y s h a d in g is u n la b e le d c o n t r o l. (C ) M C a t 1 x 10s/m L w e r e s tim u la te d w i t h IL-33 (1 0 n g /m L ) f o r 1, 2, o r 3 d in re p lic a te s . T h e re la tiv e g e n e e x p re s s io n o f
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in d e p e n d e n t e x p e rim e n ts . (D ) T w e n t y - f o u r h o u rs a f t e r siR N A
t r a n s fe c t io n , M C w e r e tr e a t e d w i t h
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p re s e n c e o r a b s e n c e o f SCF f o r 3 d . T h e f o ld in c re a s e in a p o p to s is w a s c a l c u la te d b y n o r m a liz in g e a c h g r o u p t o its b a s e lin e in t h e p re s e n c e o f SCF. T h e d a ta p r e s e n te d a re c o m b in e d f r o m th r e e in d e p e n d e n t e x p e rim e n ts . 0 .0 5 ;
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derived MC (BMMC) (20-23). IL-10 can either promote or im pair survival depending on the type of MC studied (21, 22). r e c e p to r -r e la te d p r o te in ST2 a t h ig h le v e l, a n d th is IL-1 f a m ily Recently, substantial attention has focused on the role of c y to k in e b o th a c tiv a te s M C d ir e c tly a n d p rim e s t h e m t o re s p o n d to IL-33 in MC biology. A member of the IL-1 family, IL-33 is o t h e r p r o in f la m m a t o r y s ig n a ls . W h e t h e r IL -3 3 a n d ST2 p la y a ro le expressed primarily by stromal cells, such as epithelial cells, in M C s u rv iv a l re m a in s t o b e d e fin e d . In s k in -d e r iv e d h u m a n M C , endothelial cells, and fibroblasts, as well as by certain hema w e f o u n d t h a t IL -3 3 a t t e n u a t e d M C a p o p to s is w it h o u t a lte r in g topoietic cells, including MC themselves (24-26). IL-33 is an p ro life r a tio n , a n e ffe c t m e d ia te d p rin c ip a lly th r o u g h th e a n tia p o p “alarmin’’ released upon cell necrosis and also can be secreted t o t ic m o le c u le B -c e ll ly m p h o m a - X la r g e (B C L X L ). M u r i n e M C by live cells after mechanical stretching and via other incomplete d e m o n s t r a t e d a s im ila r m e c h a n is m , d e p e n d e n t e n t i r e l y o n ST2. ly defined mechanisms (27, 28). Its receptor ST2 (also called In lin e w it h th e s e o b s e rv a tio n s , S t2 ~ '~ m ic e e x h ib ite d re d u c e d IL-1 receptor-related protein 1) is highly expressed by MC. n u m b e rs o f tis s u e M C in in fla m e d a r th r itic jo in ts , in h e lm in th Acting via ST2, IL-33 participates in MC activation in murine in fe c te d in te s tin e , a n d in n o r m a l p e r ito n e u m . T o c o n fir m a n M C models of anaphylaxis and arthritis, serving both as a direct MC in trin s ic ro le f o r ST2 in v iv o , w e p e r fo r m e d p e r ito n e a l tr a n s f e r o f activator and as a factor that primes MC for enhanced cytokine W T a n d S t2 ~ '~ M C . In S t2 ~ '~ h o s ts t r e a t e d w it h IL -3 3 a n d in W T release upon subsequent stimulation by IgE and IgG (29-32). h o s ts s u b je c te d t o th io g ly c o lla te p e r ito n itis , W T M C d is p la y e d IL-33 also prominently modulates the MC phenotype, pro a c le a r s u r v iv a l a d v a n t a g e o v e r c o e n g r a f t e d S f 2 _ /~ M C . IL -3 3 moting accumulation of MC protease 6 (the murine ortholog of b lo c k a d e s p e c ific a lly a t t e n u a t e d th is s u rv iv a l a d v a n ta g e , c o n fir m human tryptase (3) in vitro and in vivo (33). in g IL -3 3 as t h e r e le v a n t ST2 lig a n d m e d ia tin g M C s u rv iv a l in v iv o . In cardiomyocytes and hepatocytes, IL-33 protects against T o g e t h e r , t h e s e d a t a r e v e a l a c e ll-in tr in s ic r o le f o r t h e IL -3 3 /S T 2 apoptosis (34, 35), raising the possibility of an analogous effect in a x is in t h e r e g u la t io n o f a p o p to s is in M C , i d e n t i f y i n g t h e r e b y MC. Improved in vitro survival has been observed in human a p r e v io u s ly u n a p p r e c ia t e d p a t h w a y s u p p o r t in g e x p a n s io n o f umbilical cord blood-derived MC exposed to IL-33 and in cul t h e M C p o p u la t io n w i t h i n f la m m a t io n . tured murine MC under certain conditions, but this effect is incompletely characterized, and its relevance in vivo remains a r th ritis | h e lm in th in fe c tio n unknown (36, 37). In the present study, we show that IL-33 promotes the survival of both human and murine MC, mediated ast cells (MC) are tissue-resident effector cells that con principally by up-regulation of the antiapoptotic factor BCLXL. tribute both to innate and adaptive immunity (1). MC help In vivo, IL-33 and ST2 play a nonredundant role in promoting defend against bacterial and helminthic infection (2, 3) and play a role in tissue remodeling (4, 5). MC can also contribute to Significance a variety of allergic and nonallergic inflammatory diseases, such as anaphylaxis, atopic dermatitis, asthma, inflammatory arthritis, M a s t cells (M C ) a r e lo n g -liv e d cells t h a t a c c u m u la te in in fla m e d psoriasis, and multiple sclerosis (6, 7). Under many of these tis s u e s . IL -3 3 h a s b e e n c h a r a c te riz e d e x te n s iv e ly as a M C a c ti conditions, the number of MC in affected tissues increases by v a t o r , b u t a c o n t r ib u t io n o f IL -3 3 a n d its r e c e p to r IL-1 re c e p to r tenfold or more, amplifying the contribution of MC-derived r e la t e d p r o te in S T2 t o M C s u r v iv a l r e m a in s u n a p p r e c ia t e d . mediators to ongoing inflammation (6-8). Regulation of the MC H e r e , w e s h o w t h a t IL -3 3 a t t e n u a t e s a p o p to s is o f h u m a n a n d population is poorly understood, and therapeutic intervention to m u r in e M C , p r in c ip a lly v ia t h e a n t ia p o p t o t ic m o le c u le B-cell limit MC accumulation potentially could attenuate injury asso ly m p h o m a -X la rg e (B C LX L). In v iv o , IL -3 3 a n d ST2 ca n c o n fe r ciated with inflammatory diseases (9, 10). a c e ll-in trin s ic s u rv iv a l a d v a n ta g e t o m u r in e M C , p a r tic u la r ly in The survival of mature MC in tissues depends on signals from t h e c o n t e x t o f in f la m m a tio n . T h e s e re s u lts id e n t if y t h e IL -3 3 / neighboring cells (11, 12). The single most important mediator in ST2 a x is as a n im p o r t a n t p a t h w a y s u p p o r tin g M C p e rs is te n c e this process is stem cell factor (SCF), a master regulator of MC in tis s u e s , ra is in g t h e p o s s ib ility t h a t th e r a p e u t ic t a r g e t in g proliferation, differentiation, survival, and activation. Mice with o f IL -3 3 c o u ld lim it t h e c o n t r ib u t io n o f M C t o c h ro n ic in genetic mutations affecting SCF or its receptor Kit have few or f la m m a t o r y d is ea ses . no MC in healthy or inflamed tissues, whereas activating muta tions of Kit give rise to pathologic mastocytosis (13-15). IL-3 is A u th o r contributions: J.-X.W., S.K., C.L.K., M.F.G., and P.A.N. designed research; J.-X.W., S.K., S.A., N.F., D.D., A.D., and M.F.G. perform ed research; A.D. and C.L.K. contributed another well-recognized MC growth factor (16). SCF/Kit and new reagents/analytic tools; J.-X.W., S.K., M.F.G., and P.A.N. analyzed data; and J.-X.W., IL-3 interface with several antiapoptotic pathways, up-regulating S.K., and P.A.N. w rote th e paper. the antiapoptotic protein B-cell lymphoma-2 (BCL-2) and down The authors declare no conflict o f interest. regulating the proapoptotic protein Bim (14, 17-19). Aside from This article is a PNAS Direct Submission. C.G. is a guest e d ito r invited by th e Editorial SCF/Kit and IL-3, other cytokines have been reported to support Board. MC survival, but their effects appear to be restricted to specific 1J.-X.W. and S.K. contributed equally to this w ork. MC subtypes. IL-4 promotes survival of intestinal MC via both 2To w hom correspondence should be addressed. E-mail: pnigrovic@ partners.org. BCL-2 and B-cell Iymphoma-X large (BCLXL) but may suppress This article contains supporting inform a tio n online a t www .pnas.org/lookup/suppl/doi: 10. survival in human cord blood MC as well as murine bone marrow1073/pnas. 1404182111/-/DCSupplemental. c h ro n ic a lly in fla m e d tissu e s. M C e x p re s s t h e IL -3 3 r e c e p to r IL-1
www.pnas.org/cgi/doi/10.1073/pnas.1404182111
PNAS
| July 1 5 ,2 0 1 4
| vol. 111
| no. 28
| 102 81-10286
IMMUNOLOGY AND INFLAMMATION
M
than that of siRNA #1 (~60%) (Fig. SL4). Under SCF culture conditions, potent knockdown of either gene increased apoptosis by 1.4- to 1.7-fold (Fig. SIB) (16). The protective effect of IL-33 was almost abolished by silencing BCLXL, whereas BCL-2 knock down had a minor impact (Fig. ID). Taken together, these results indicate that IL-33 protects human MC from apoptosis primarily through expression of BCLXL. T h e IL-33 R e c e p to r ST2 Is R e q u ire d f o r th e P ro s u rv iv a l E ffe c t o f IL-33.
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w e r e tr e a t e d w i t h o r w i t h o u t IL-33 (1 0 n g /m L ) in t h e p re s e n c e o r a b s e n c e o f SCF f o r 3 d . M C a p o p to s is w a s assessed b y f l o w c y to m e tr y . (B) C ell d iv is io n w a s m e a s u re d b y CFSE d ilu t io n assay in liv e M C (A n n e x in V “ P r ) w i t h o r w i t h o u t IL-33 in t h e a b s e n c e o f SCF f o r 3 d . G ra y s h a d in g is u n la b e le d c o n t r o l. (C ) M C a t 1 x 10s/m L w e r e s tim u la te d w i t h IL-33 (1 0 n g /m L ) f o r 1, 2, o r 3 d in re p lic a te s . T h e re la tiv e g e n e e x p re s s io n o f
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in d e p e n d e n t e x p e rim e n ts . (D ) T w e n t y - f o u r h o u rs a f t e r siR N A
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