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1 1 E 1 2 1 1 1 oX oX ? 1 1 1 [ 2 2 2 2 a 1 1 1 1 1 1 1 1 1 : HMGB1-promoted and TLR2/4-dependent NK cell maturation and activation take part in rotavirus-induced murine biliary atresia
Yinrong Qiu*1, Jixin Yang*1, Wenmei Wang1, Wentao Zhao1, Fei Peng1, Ying Xiang2, Gang Chen2, Tao Chen3, Chengwei Chai1, Shuaiyu Zheng1, Daniel J Watkins4, Jiexiong Feng1
* These authors contribute equally to this work
1 Department of Pediatric Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China, 430030
2 Institute of Organ Transplantation, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China, 430030
3 Department of Infectious Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China, 430030
4 Department of Surgery, Wayne State University, Detroit, Michigan, USA, 48201
This work is supported by National Natural Science Foundation of China (Grant No. 30672195, 81070284 and 81270441)
None of the authors have any conflicts of interest.
Corresponding author:
Jiexiong Feng, MD, PhD
Department of Pediatric Surgery,
Tongji Hospital of Tongji Medical College,
Huazhong University of Science and Technology,
Wuhan, China, 430030
fengjiexiong@126.com
Short Title: Dysfunction of NK cells in Murine Biliary Atresia
Key words: Biliary atresia; Rotavirus; Natural killer cells; Toll-like receptors; HMGB1
ABSTRACT
Recent studies show that NK cells play important roles in murine biliary atresia (BA), and a temporary immunological gap exists in this disease. In this study, we found high-mobility group box-1 (HMGB1) and TLRs were overexpressed in human and rotavirus-induced murine BA. The overexpressed HMGB1 released from the nuclei of rotavirus-infected cholangiocytes, as well as macrophages, activated hepatic NK cells via HMGB1-TLRs-MAPK signaling pathways. Immature NK cells had low cytotoxicity on rotavirus-injured cholangiocytes due to low expression of TLRs, which caused persistent rotavirus infection in bile ducts. HMGB1 up-regulated the levels of TLRs of NK cells and promoted NK cell activation in an age-dependent fashion. As NK cells gained increasing activation as mice age, they gained increasing cytotoxicity on rotavirus-infected cholangiocytes, which finally caused BA. Adult NK cells eliminated rotavirus-infected cholangiocytes shortly after infection, which prevented persistent rotavirus infection in bile ducts. Moreover, adoptive transfer of mature NK cells prior to rotavirus infection decreased the incidence of BA in newborn mice. Thus, the dysfunction of newborn NK cells may, in part, participate in the immunological gap in the development of rotavirus induced murine BA.
AUTHOR SUMMARY
Biliary atresia (BA) is the most common precipitating factor for liver transplantation in infants. BA is caused by the obstruction of hepatic bile ducts, leading to progressive obstructive jaundice and liver fibrosis. A well-recognized theory is that rotavirus injures biliary epithelia in a mouse model of BA, followed by attack of immunocytes, such as NK cells. We performed this research to investigate whether maturation and activation of NK cells take part in the development of BA. We identified that rotavirus induced HMGB1 release from injured bile ducts. HMGB1 induced NK cell activation in an age-dependent fashion via HMGB1-TLRs-MAPK signaling pathways. Newborn NK cells were unable to eliminate rotavirus-infected cholangiocytes, which caused persistent biliary infection; maturated NK cells were activated gradually and caused persistent biliary injury, which finally led to BA. We identify HMGB1 as an important pro-inflammatory initiator and a critical inducer for maturation of NK cells in the development of BA. HMGB1-induced activation of NK cells may, in part, plays crucial roles in the development of murine BA. Novel therapies targeting HMGB1 or TLRs in patients with BA may be applied in the future to decrease the activity of NK cells in order to inhibit the progression of BA.
INTRODUCTION
Biliary atresia (BA), which is the most common precipitating factor leading to liver transplantation in infants ADDIN EN.CITE de Carvalho20071[1]1117de Carvalho, E.Ivantes, C. A.Bezerra, J. A.Universidade de Brasilia, Brasilia, DF, Brazil. elisacarvalho@terra.com.brExtrahepatic biliary atresia: current concepts and future directionsJ Pediatr (Rio J)105-208322007/04/12*Biliary Atresia/diagnosis/etiology/therapyChildForecastingHumansLiver TransplantationPortoenterostomy, HepaticPrognosis2007Mar-Apr0021-7557 (Print)
0021-7557 (Linking)17426869http://www.ncbi.nlm.nih.gov/pubmed/1742686910.2223/JPED.1608eng[ HYPERLINK \l "_ENREF_1" \o "de Carvalho, 2007 #1" 1], is a common neonatal cholangiopathy that leads to progressive hepatobiliary injury ADDIN EN.CITE ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_2" \o "Sokol, 2007 #2" 2]. BA has been recognized as a virus-induced autoimmune disease ADDIN EN.CITE ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_3" \o "Mack, 2007 #3" 3, HYPERLINK \l "_ENREF_4" \o "Feng, 2008 #5" 4], in which infection by viruses, especially rotavirus, is often considered as the initiator in the pathogenesis ADDIN EN.CITE ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_5" \o "Petersen, 1997 #6" 5]. In murine BA, rotavirus infection is followed by activation of lymphocytes and secretion of inflammatory cytokines ADDIN EN.CITE ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_6" \o "Li, 2011 #11" 6, HYPERLINK \l "_ENREF_7" \o "Shivakumar, 2004 #10" 7] targeting extrahepatic bile ducts. We have previously shown the importance of leukocyte antigen-DR ADDIN EN.CITE ADDIN EN.CITE.DATA [ H Y P E R L I N K \ l " _ E N R E F _ 8 " \ o " F e n g , 2 0 0 4 # 1 2 " 8 ] a n d o s t e o p o n t i n A D D I N E N . C I T E A D D I N E N . C I T E . D A T A [ H Y P E R L I N K \ l " _ E N R E F _ 9 " \ o " H u a n g , 2 0 0 8 # 1 3 " 9 ] i n h u m a n B A . I n a n i m a l s t u d i e s , w e h a v e d e m o n s t r a t e d t h a t N F - B r e g u l a t e s r h e s u s r o t a v i rus (RRV)-induced BA ADDIN EN.CITE ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_10" \o "Feng, 2005 #15" 10, HYPERLINK \l "_ENREF_11" \o "Huang, 2010 #16" 11]. We have recently reported that rotavirus NSP4 is a crucial immunogen in BA ADDIN EN.CITE ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_12" \o "Feng, 2011 #17" 12]. Our previous findings have reinforced the notion that BA is a virus-induced and immune system mediated disease ADDIN EN.CITE Feng20085[4]5517Feng, J.Huang, L. The virus infection and biliary atresiaCurr Pediatr Rev164-842008[ HYPERLINK \l "_ENREF_4" \o "Feng, 2008 #5" 4].
It is reported that high-mobility group box-1 (HMGB1) protein, which is a nuclear factor released extracellularly from immune cells or injured non-immune cells ADDIN EN.CITE ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_13" \o "Xu, 2010 #29" 13] and acts as an important mediator of various inflammatory responses, is demonstrated to interact with TLR2 and TLR4 ADDIN EN.CITE ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_14" \o "Park, 2006 #30" 14]. However, it is poorly understood whether HMGB1 interacts with TLR2 and TLR4 to induce murine BA.
Moreover, there is a temporary immunological gap in murine BA reported by Czech-Schmidt et al ADDIN EN.CITE ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_15" \o "Czech-Schmidt, 2001 #18" 15]. In their study, when infected by RRV 12 hours after birth, the incidence of BA was 86% and a mortality of 100%. When the newborn mice were infected 24 h postpartum, 65% of newborn mice developed murine BA with a mortality of 69%; whereas no adult mice infected by RRV acquired BA ADDIN EN.CITE ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_15" \o "Czech-Schmidt, 2001 #18" 15]. In this model, various immunocytes are shown to participate in development of BA ADDIN EN.CITE ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_6" \o "Li, 2011 #11" 6, HYPERLINK \l "_ENREF_7" \o "Shivakumar, 2004 #10" 7] and some studies have demonstrated the importance of NK cells in targeting cholangiocytes after viral infection ADDIN EN.CITE ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_16" \o "Shivakumar, 2009 #20" 16, HYPERLINK \l "_ENREF_17" \o "Miethke, 2010 #53" 17]. However, no study has yet investigated the roles of NK cell maturation and activation in the immunological gap of murine BA.
In the present study, we found that the expression of HMGB1 is increased in human/murine BA, and the overexpressed HMGB1 is released from injured cholangiocytes and macrophages, which activates NK cells via activation of HMGB1-TLRs-MAPK signaling pathways. Immature NK cells are incapable of eliminating RRV-infected cholangiocytes in neonates, which causes persistent RRV infection in bile ducts. HMGB1 promotes maturation of NK cells as mice age, leading to an increased and persistent immune response in cholangiocytes, which induces BA. On the other hand, the activation of NK cells of adult mice is increased and their mature NK cells eliminate RRV-infected cholangiocytes shortly after infection. Therefore, in RRV-infected adult mice, the biliary tracts are not damaged and BA does not develop. Thus, immature NK cells take part in the immunological gap in the development of RRV-induced murine BA.
RESULTS
Expression of HMGB1, TLR2 and TLR4 are increased in livers of patients with BA
HMGB1 is shown to be increased during inflammation ADDIN EN.CITE ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_13" \o "Xu, 2010 #29" 13], and its receptors TLR2/TLR4 are demonstrated to be important in various types of obstructive cholangiopathy ADDIN EN.CITE Miranda-Diaz201123[18]232317Miranda-Diaz, A. G.Alonso-Martinez, H.Hernandez-Ojeda, J.Arias-Carvajal, O.Rodriguez-Carrizalez, A. D.Roman-Pintos, L. M.Centro Universitario de Ciencias de la Salud, Universidad de Guadalajara, Guadalajara, 44340 JAL, Mexico.Toll-like receptors in secondary obstructive cholangiopathyGastroenterol Res Pract26509320112011/11/2520111687-630X (Electronic)
1687-6121 (Linking)22114589http://www.ncbi.nlm.nih.gov/pubmed/22114589320572310.1155/2011/265093eng[ HYPERLINK \l "_ENREF_18" \o "Miranda-Diaz, 2011 #23" 18]. BA is a typical obstructive biliary disease with inflammation of extra and intra-hepatic bile ducts, so we investigated whether HMGB1 and TLRs were increased in livers from patients with BA. Livers derived from patients with congenital dilation of the bile duct (CDB), which were not ideal controls but with relatively minor liver lesion and easy to acquire due to ethical considerations ADDIN EN.CITE Funaki199831[19]313117Funaki, N.Sasano, H.Shizawa, S.Nio, M.Iwami, D.Ohi, R.Nagura, H.Department of Pathology, Tohoku University School of Medicine, Sendai, Japan.Apoptosis and cell proliferation in biliary atresiaJ Pathol429-3318641999/04/21*ApoptosisBile Ducts, Extrahepatic/pathologyBiliary Atresia/*pathologyCell DivisionDilatation, Pathologic/congenital/pathologyFemaleHumansIn Situ Nick-End LabelingInfantInfant, NewbornKi-67 Antigen/metabolismLiver/cytology/pathologyMale1998Dec0022-3417 (Print)
0022-3417 (Linking)10209494http://www.ncbi.nlm.nih.gov/pubmed/1020949410.1002/(SICI)1096-9896(199812)186:4<429::AID-PATH195>3.0.CO;2-6 [pii]
10.1002/(SICI)1096-9896(199812)186:4<429::AID-PATH195>3.0.CO;2-6eng[ HYPERLINK \l "_ENREF_19" \o "Funaki, 1998 #31" 19], were used as controls to compare to BA livers. The clinical data of patients were summarized in Table S1 and described in Supplemental Protocols. Increased staining of HMGB1 in the periductal area and increased staining of TLR2 and TLR4 in the periductal area and infiltrated cells were noted in liver tissues of patients with BA compared to patients with CDB (Fig 1A). Western blotting analyses showed significantly increased protein levels of HMGB1, TLR2 and TLR4 (all, p<0.05) in liver tissues from patients with BA compared to patients with CDB. The realtime RT-PCR showed the livers from patients with BA had significantly increased mRNA levels of HMGB1, TLR2 and TLR4 (all, p<0.05) compared to patients with CDB (Fig 1B and 1C).
Increased expression of HMGB1, TLR2 and TLR4 in the livers is observed in RRV-induced murine BA
To investigate whether murine BA has similar increases of HMGB1, TLR2 and TLR4 as human BA, we next detected their expression in livers of mice on day 7 after RRV-infection. The immunochemical staining showed that non-RRV infected wildtype (WT) C57BL/6 (B6) mouse pups (left panels in Fig 1D) had low expression of HMGB1 mainly localized inside the nuclei of cholangiocytes. In pups exposed to RRV infection, increased HMGB1 staining was found not only in the mucosal layer of the cholangiocytes, but also in cells in the periductal areas. Realtime RT-PCR showed an approximately 7-fold increase of HMGB1 mRNA in the livers of RRV-infected pups compared to pups without RRV challenge (p<0.01) (Fig 1E). Increased staining of TLR2 and TLR4 was observed in the livers of pups exposed to RRV infection (middle and right panels in Fig 1D). Realtime RT-PCR showed that livers from RRV infected mice had a 4- to 6-fold increase of TLR2 and TLR4 compared to non-RRV challenged mice (both, p<0.01) (Fig 1F and 1G). These findings confirmed that human BA and RRV-induced murine BA have similar increases of HMGB1, TLR2 and TLR4, indicating that murine BA, in part, mimics the pathophysiological changes of human BA.
Rotavirus infection induces release of HMGB1 from cholangiocytes and macrophages
Now that we have confirmed the similarly increase of HMGB1 is noted bile ducts and periductal area both in human and murine BA, we designed in vitro studies to investigate whether RRV-infected cholangiocytes or macrophages release HMGB1. Immunofluorescent staining showed that HMGB1 was localized in nuclei of cholangiocytes at a 0 hour time point of RRV infection. HMGB1 release from nuclei began 12 hours after RRV incubation, and a large amount of nuclear HMGB1 in the nuclei was released extracellularly at 24 and 36 hours after RRV infection (Fig 2A). Nuclear HMGB1 staining weakened starting from 24 hours after RRV infection, while HMGB1 in non-infected cholangiocytes was localized in the nuclei at all time points (Fig 2B). The mRNA level of HMGB1 in cholangiocytes was increased significantly at 12 hours, 24 hours and 36 hours (all, p<0.01) after RRV infection compared to control groups (Fig 2C). Unlike the mRNA levels, there was no significant difference in the protein level of HMGB1 in the culture medium between the infected and control cholangiocytes at the 12 hour time point (p>0.05), whereas the concentration of extracellular HMGB1 protein was increased significantly in the RRV-infected group compared to the control at the 24 hour and the 36 hour (both, p<0.01) time points. Although synthesis and release of HMGB1 were both increased, the percentage increase of HMGB1 release was higher than that of HMGB1 synthesis at 24 hour (84.4% vs. 47.4%) and 36 hour (119.8% vs. 54.0%) time points. This may cause decreased staining of HMGB1 in nuclei at 24 hour and 36 hour time points (Fig 2D). In addition, 24 hours after RRV infection, both newborn and adult macrophages have increased release of HMGB1 compared to their controls respectively (both, p<0.05), however, there was no significant difference in the level of HMGB1 between the newborn and adult macrophages after RRV infection (p>0.05) (Fig S1). These findings indicate that an important component of increased HMGB1 in RRV-induced murine BA may be the nuclei of RRV-infected cholangiocytes, as well as other RRV infected immunocytes such as macrophages; the increase of HMGB1 mRNA synthesis in cholangiocytes starts at an early time point, followed by persistent release of HMGB1 from 24 hours up to 36 hours after RRV infection.
HMGB1 induces activation of NK cells as mice age from 1 day old mice to adult mice
Our current studies have shown that an increased level of HMGB1 is observed in the development of BA, and NK cells are shown to play important roles in this disease ADDIN EN.CITE ADDIN EN.CITE.DATA [ HYPERLINK \l "_ENREF_16" \o "Shivakumar, 2009 #20" 16, HYPERLINK \l "_ENREF_17" \o "Miethke, 2010 #53" 17], we next investigated whether HMGB1 directly activates NK cells in a time-dependent fashion. The adult mice h a d t h e h i g h e s t e x p r e s s i o n o f C D 6 9 , T N F - a n d I F N - c o m p a r e d t o t h o s e i n t h e o t h e r 2 a g e g r o u p s . W e f o u n d s i g n i f i c a n t l y i n c r e a s e d e x p r e s s i o n o f C D 6 9 , T N F - a n d I F N - i n N K c e l l s d e r i v e d f r o m a l l a g e g r o u p s i n H M G B 1 - s t i m u l a t e d N K c e l l s c o m p a r e d t o n o n - s t i m u l a t e d N K c e l l s ( p <