Targeting Extracellular Cyclophilins Ameliorates Disease Progression in Experimental Biliary Atresia
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Biliary atresia (BA) is a devastating liver disease of unknown etiology affecting children generally within the first 3 months of life. The disease is manifested by inflammation and subsequent obstruction of the extrahepatic bile ducts, fibrosis and liver failure. The mechanisms responsible for disease pathogenesis are not fully understood, but a number of factors controlled by the SMAD signaling pathway have been implicated. In this study, we investigated the role of a known proinflammatory factor, extracellular cyclophilin A (CypA), in the pathogenesis of biliary atresia using the rhesus rotavirus (RRV) murine model. We used a unique cyclosporine A derivative, MM284, which does not enter cells and therefore inactivates exclusively extracellular cyclophilins, as a potential treatment. We demonstrated that levels of CypA in plasma of RRV-infected mice were increased significantly, and that treatment of mice with MM284 prior to or one day after disease initiation by RRV infection significantly improved the status of mice with experimental BA: weight gain was restored, bilirubinuria was abrogated, liver infiltration by inflammatory cells was reduced and activation of the SMAD pathway and SMAD-controlled fibrosis mediators and tissue inhibitor of metalloproteinases (TIMP)-4 and matrix metalloproteinase (MMP)-7 was alleviated. Furthermore, treatment of human hepatic stellate cells with recombinant cyclophilin recapitulated SMAD2/3 activation, which was also suppressed by MM284 treatment. Our data provide the first evidence that extracellular cyclophilins activate the SMAD pathway and promote inflammation in experimental BA, and suggest that MM284 may be a promising therapeutic agent for treating BA and possibly other intrahepatic chronic disorders.
Biliary atresia (BA) is a devastating liver disease in children that results in obstruction of the biliary system and, without treatment, death within two years of birth due to hepatic cirrhosis. The incidence of BA is quite rare; it is diagnosed in approximately one in 10,000 children in the first three months of life. The pathophysiology of BA is inflammation and obstruction of the extrahepatic bile ducts that results in fibrosis, subsequent cirrhosis and eventual liver failure. Surgical treatment via portoenterostomy results in only ∼60% transplant-free survival 2 years after surgery (1). Since the exact cause of the disease is unknown and medical and surgical treatments remain suboptimal, clarification of the mechanisms involved in initial induction and progression of biliary inflammation and identification of potential targets for therapeutic intervention remain essential for new treatment strategies.
The murine model of BA in neonatal mice infected with rhesus rotavirus (RRV) has been used to study the pathogenesis of the disease (2,3). In our early studies using this model, we demonstrated increased mRNA expression of the matrix metalloproteinases (MMPs) in liver tissue samples of infected mice (4). We had initially suspected that these genes may be upregulated via the transforming growth factor (TGF)β pathway, as we had also seen perturbations in mRNA expression of tissue inhibitor of metalloproteinases (TIMP)-1 and TIMP-4 as well as plasminogen activator inhibitor-1 (PAI-1), which are all regulated by TGFβ. However, several experiments utilizing anti-TGF strategies, comprised mainly of antibody blockade of TGF and its pathway members, failed to demonstrate any effect in this animal model (data not shown). Therefore, we began to explore alternative mechanisms that could be responsible for BA-associated inflammation.
Studies of tumorigenesis have shown that increased levels of MMPs are detected in stromal fibroblasts, endothelial cells and in the tumor cells themselves in response to activation of extracellular matrix metalloproteinase inducer (EMM-PRIN) or CD147 (5). CD147 is a type I transmembrane glycoprotein, a member of the immunoglobulin super-family, that is expressed by a wide array of cell types, including epithelial, endothelial and hematopoietic cells (6). CD147 is a signaling receptor for extracellular cyclophilins (Cyp) A and B (7,8), and recent publications have identified the CD147 signaling pathway induced by extracellular cyclophilins as a triggering mechanism regulating MMP expression and inflammation in atherosclerosis, rheumatoid arthritis, allergic and chronic lung disease (9, 10, 11, 12, 13, 14). Thus, we hypothesized that CD147 activation by extracellular cyclophilin may play a role in experimental BA as well.
Previous studies showed that the interaction of extracellular CypA with CD147 induces activation of extracellular signalregulated kinase (ERK) pathway and a downstream increase in the expression of interleukins, MMPs and CD147 itself, resulting in the progression of inflammation (9,15,16). Thus, blockade of extracellular cyclophilin-CD147 interactions may have a potential to reduce inflammation and infiltration of inflammatory cells into infected tissues. In fact, studies of serum inflammatory cytokine profiles demonstrated that treatment of acute systemic vasculitis (Kawasaki disease) with cyclosporine (a known cyclophilin inhibitor) resulted in downregulation of cyclophilin-induced pathways, including CD147-mediated inflammation (17). Therefore, specific targeting of extracellular cyclophilins may be a valid therapeutic strategy to inhibit CD147-dependent pathways. MM284 is a nonimmunosuppressive cell-impermeable cyclosporine derivative that does not penetrate the plasma membrane and, therefore, cannot interact with intracellular cyclophilins and mediate intracellular immunosuppressive activity; thus MM284 targets only the extracellular pool of cyclophilins (12,18). We hypothesized that the liver inflammation associated with BA may be induced or activated by the interaction of extracellular CypA with CD147 located at the plasma membrane of hepatocytes and hepatic stellate cells, and blocking this interaction by MM284 would inhibit this inflammatory response and subsequent liver inflammation and, potentially, fibrosis.
Materials and Methods
Murine Model of Biliary Atresia
Pregnant time-dated BALB/c mice (Charles River Labs) were kept with one animal per cage with free access to water and the standard laboratory diet. After spontaneous vaginal delivery, the newborn mice were randomly divided into four groups. During the first 24 h of life, mice in the control group (n = 10) received an intraperitoneal injection of 15% Cremophor EL (Sigma-Aldrich) or saline. The initial set of animals in this group (n = 5) was injected with 15% Cremophor EL, and no effect of the Cremophor EL on mouse weight or bilirubinuria was confirmed by comparing these mice to mice receiving saline alone (n = 5) (data not shown). In the second group (RRV group, n = 23), 1.5 × 106 fluorescence forming units of rhesus rotavirus (RRV) was administered, and 15% Cremophor EL was injected on d 0, 2, 4, 6, 8, 10 and 12. The third group (MM284 + RRV group, n = 20 total) received RRV plus 20 mg/kg of MM284 in 15% Cremophor EL. The fourth group (MM284 group, n = 5) received 20 mg/kg of MM284 in 15% Cremophor EL only. The animal data were collected from three separate animal trials to account for the variability in the RRV model. We used two different schedules for MM284 treatment for group 3. In the first set of experiments (n = 15), animals received the drug on d 0, 2, 4, 6, 8, 10 and 12. This represented initiation of MM284 treatment prior to RRV administration and consequently prior to the onset of disease (pretreatment). In the second set of experiments (n = 5), treatment with MM284 was initiated on d 1 after RRV treatment. The treated mice were kept with their mothers, maintained in normal environment and housed in a room with a standard 12-h dark-light cycle. Subsequent experimental procedures (such as clinical phenotyping, subcutaneous saline injection and organ harvest) were performed as previously described (4). Liver specimens were harvested for protein and RNA isolation, as well as histological and immunohistochemical analysis. All procedures were approved by the Children’s National Medical Center Institutional Animal Care and Use Committee (IACUC).
RNA Isolation and Real-time Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
Primers for proinflammatory cytokines
Forward primer (5′–3′)
Reverse primer (5′–3′)
Primers for fibrosis mediators
Forward primer (5′–3′)
Reverse primer (5′–3′)
Enzyme-Linked Immunosorbent Assay (ELISA)
The concentration of phosphorylated SMAD2 was determined using the commercially available PathScan P-SMAD2 Sandwich ELISA kit (Cell Signaling Technology). Liver samples were homogenized according to the manufacturer’s instructions in lysis buffer using homogenizer FastPrep-24 (MP Biomedicals). Subsequent manipulations were performed as described earlier (19). The concentration of murine CypA in plasma was determined using ELISA kits from MyBioSource following the manufacturer’s instructions.
MM284, a side-chain modification in the 1-position of cyclosporine A (CsA) was prepared according to published procedures (20). For injection, the powder was first dissolved in 100% ethanol (Sigma-Aldrich) and then diluted in sterile 15% solution of Cremophor EL (Sigma-Aldrich) in PBS.
Human recombinant cyclophilin A was an Enzo Life Science product.
Human hepatic stellate cells (HSC) were a product of ScienCell Research Laboratories. Cells were cultured in the special stellate cell medium supplemented with 1% stellate cell grow supplement, 2% fetal bovine serum and penicillin-streptomycin solution (100 U/mL and 100 µg/mL, respectively). HSC cells were cultured in poly-d-lysine-coated 75-cm2 culture flasks (Becton Dickinson Labware) at 37°C and 5% CO2/95% air atmosphere. Cultured HSCs were treated with recombinant CypA (800 ng/mL) with or without MM284 (400 ng/mL) and then incubated for 72 h. The dose of CypA used in this experiment was about 15-fold higher than the level measured in the plasma of BA mice to account for higher concentration of cyclophilin at the sites of inflammation; this concentration is similar to CypA levels in the serum of patients with systemic inflammation observed in sepsis (22).
Presence of CypA in RRV-Treated Mice
MM284 Treatment Restores Weight Gain and Prevents Bilirubinuria in BA Mice
We have previously shown that bilirubinuria correlates well with biliary obstruction and with histologic BA in the experimental model of BA (4). In the current study, no animals treated with saline or Cremophor EL alone without RRV infection displayed bilirubinuria on a urine dipstick. In animals infected with RRV but not receiving MM284, 21 of the 23 were positive for bilirubinuria on d 14 (Figure 2C). In contrast, only 8 out of 20 MM284-treated mice were positive for bilirubinuria after RRV infection. Both the pretreatment and posttreatment groups had the same proportion of bilirubinuria-positive mice (6/15 and 2/5, respectively, p = 1.0 Fischer’s Exact test). When combining the pretreatment and posttreatment groups and comparing them to untreated animals, the chances that the difference between MM284-treated and control animals was due to chance alone were insignificant (p < 0.001, chi-square test).
MM284 Ameliorates the Inflammatory Response in BA Mice
We previously demonstrated a dramatic increase of mRNA expression of certain mediators of inflammation and fibrosis, such as TIMP-1, TIMP-4, PAI-1, MMP-7 and MMP-9 in the mouse model of BA (4). In the present study, we found that treatment with MM284 significantly reduced expression of two of these mediators, TIMP-4 and MMP-7, in the liver homogenates of mice with RRV-induced BA. Pretreatment with MM284 resulted in an over six-fold (6.6 ± 1.1, p < 0.001) reduction of TIMP-4 mRNA in the liver relative to untreated animals on d 14 (Figure 3B), and posttreatment with MM284 decreased the TIMP-4 mRNA approximately five-fold (5.4 ± 1.12, p < 0.01) (Figure 3C). An even more pronounced effect was observed on MMP-7 mRNA expression: it was reduced about ten-fold on d 14 postinfection in both mice pretreated and posttreated with MM284 (10.7 ± 2.2, p < 0.001 and 9.9 ± 0.7, p < 0.01 respectively, relative to untreated mice) (Figures 3D, E). However, TIMP-4 and MMP-7 mRNA expression levels in MM284-treated animals were still higher than those in control animals. Quantitative RT-PCR did not detect significant differences in TIMP-1, PAI-1 and MMP-9 expression, although there was a trend toward decreased expression of these mediators as well (data not shown).
MM284 Reduces Interleukin (IL)-6 mRNA Expression in the Liver of BA Mice
IL-6 is a multifunctional cytokine that is implicated in the pathogenesis of multiple diseases associated with inflammation, including inflammatory liver diseases, and is known to be secreted by macrophages in response to extracellular cyclophilin (24,25). Thus, we next investigated whether MM284 had an effect on IL-6 production in our model. RRV infection increased IL-6 mRNA expression five-fold relative to control mice 14 d after RRV infection (5.4 ± 0.3, p < 0.05); pretreatment with MM284 significantly abrogated this elevated expression (1.65 ± 0.5, p < 0.05 relative to untreated infected mice) (Figure 3F). In the experiment with MM284 posttreatment, RRV increased IL-6 mRNA expression approximately four-fold relative to the uninfected group (4.44 ± 0.2, p < 0.01) and MM284 treatment reduced IL-6 to the level similar to that in the control group (1.0 ± 0.4) (Figure 3G).
MM284 Reduces SMAD2 Activation
SMAD activation is a previously unreported action of extracellular cyclophilins. To confirm this finding, we analyzed the effect of recombinant human CypA on the inflammatory signaling pathways in human hepatic stellate cells (HSCs), which are known contributors to the pathogenesis of fibrotic liver diseases (27,28). Similar to the findings in the BA mice, lysates from CypA-treated cells demonstrated a 1.5-fold increase in SMAD2/3 phosphorylation relative to untreated cells, and a complete absence of the increased SMAD2/3 phosphorylation when the cells were treated with CypA in combination with MM284 (Figure 4B). Importantly, sensitivity to MM284 indicates that the effect of recombinant CypA was not due to possible endotoxin contamination but was mediated by a cyclosporine-sensitive interaction (29).
Increased levels of extracellular cyclophilins (mostly CypA) have been documented in many inflammatory diseases, and their role in disease pathogenesis has been verified in a variety of animal models of human diseases, including rheumatoid arthritis, sepsis, asthma, atherosclerosis and a number of viral infections (33,34). Similarly, we reported a marked inhibitory effect of an extracellularly restricted inhibitor of the peptidyl prolyl isomerase activity of cyclophilins, the highly branched CsA-like molecule MM218, on the inflammatory response in a mouse model of allergic lung inflammation (13). Furthermore, a smaller molecule member of this type of cell-impermeable cyclosporine derivatives, MM284, inhibited the recruitment of leukocytes during inflammation in a mouse model of experimentally induced peritonitis and delayed-type hypersensitivity reaction (21). Thus, we hypothesized that MM284 may provide a benefit in the experimental model of BA comprised of neonatal mice infected with RRV.
In this study we demonstrated that MM284 reduced inflammation and phenotypic signs of disease in the RRV-induced mouse model of BA. Given that interaction between virus-incorporated cyclophilin and CD147 has been implicated in infection by HIV-1 and measles (35,36), we also evaluated a group of mice which were infected with RRV first and then treated with MM284 starting 24 h after RRV infection to exclude the possibility that MM284 prevents RRV infection of the animal directly. The fact that both pretreatment and posttreatment with MM284 had similar effects on disease manifestation (less bilirubinuria, improved weight gain) indicates that the drug inhibited the inflammatory response after RRV infection rather than RRV infection itself. We found that MM284 administration also reduced mRNA expression of a number of proinflammatory mediators downstream from SMAD2 activation, which was also inhibited, suggesting that extracellular cyclophilin plays a role in the inflammation associated with this model of BA. Given that the major signaling receptor for extracellular cyclophilins is CD147 (30), our data suggest that interaction between extracellular cyclophilins and CD147 plays a significant role in inflammation and hepatic pathology in experimental BA. Indeed, several reports have demonstrated activation of the CD147-induced signaling pathways by CypA circulating in extracellular media in a number of inflammatory diseases (31). CypA has been shown to be secreted by different types of cells in response to inflammatory stimuli and oxidative stress (32, 33, 34, 35, 36). While the cells responsible for secretion of extracellular cyclophilin in our BA model have not been identified, hepatocytes may be involved as these cells have been shown to secrete CypA in response to infection by another virus, hepatitis B (37). HSCs are another type of cells that could contribute to the etiology of BA, although we did not assess whether HSCs secrete CypA in the BA model used in this study. Further experiments isolating hepatocytes and HSCs from the liver would be required to sort out the relative importance of these two cell types, however, such experiments are technically challenging in neonatal mice.
In summary, our study adds biliary atresia to the list of inflammatory diseases where extracellular cyclophilins may play a role in exacerbating disease pathology. While it would be interesting to confirm the role of CypA in experimental BA using CypA knockout mice, the specificity of the RRV model to BALB/c mice renders those experiments unfeasible at present, as the knockout mice have a different strain as their background. Our findings suggest that extracellular cyclophilins are important contributors to the liver inflammation associated with experimental BA. Most importantly, results reported here provide a validation for development of cell-impermeable cyclosporine A derivatives as therapeutics for treatment of BA or other inflammatory diseases of the liver where SMAD2 activation may be involved. As this animal model is not well suited to address fibrotic liver disease, whether or not extracellular cyclophilins in general or CypA in particular may be putative targets for therapy once fibrosis or cirrhosis has been established remains in question.
The authors declare that they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.
This work was funded in part by National Institutes of Health; Grant Number K08 DK083769.
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