Research ArticleBiliary Atresia

Large-scale proteomics identifies MMP-7 as a sentinel of epithelial injury and of biliary atresia

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Science Translational Medicine  22 Nov 2017:
Vol. 9, Issue 417, eaan8462
DOI: 10.1126/scitranslmed.aan8462

Bile duct injury marker to the rescue

Biliary atresia is a poorly understood pediatric disease, which is associated with progressive bile duct obstruction and liver injury in young infants. Early diagnosis of this condition is crucial for restoring bile flow and protecting the liver from irreversible damage, but the disease can be difficult to distinguish from more common causes of cholestasis. By performing proteomics on large sets of patient samples, Lertudomphonwanit et al. identified the serum concentration of a protein called matrix metalloproteinase-7 as a promising diagnostic marker of this disease, especially when used together with γ-glutamyltranspeptidase, a less specific marker of cholestasis. The authors also investigated the mechanistic role of matrix metalloproteinase-7 in bile ducts, supporting the biological relevance of this marker.

Abstract

Biliary atresia is a progressive infantile cholangiopathy of complex pathogenesis. Although early diagnosis and surgery are the best predictors of treatment response, current diagnostic approaches are imprecise and time-consuming. We used large-scale, quantitative serum proteomics at the time of diagnosis of biliary atresia and other cholestatic syndromes (serving as disease controls) to identify biomarkers of disease. In a discovery cohort of 70 subjects, the lead biomarker was matrix metalloproteinase-7 (MMP-7), which retained high distinguishing features for biliary atresia in two validation cohorts. Notably, the diagnostic performance reached 95% when MMP-7 was combined with γ-glutamyltranspeptidase (GGT), a marker of cholestasis. Using human tissue and an experimental model of biliary atresia, we found that MMP-7 is primarily expressed by cholangiocytes, released upon epithelial injury, and promotes the experimental disease phenotype. Thus, we propose that serum MMP-7 (alone or in combination with GGT) is a diagnostic biomarker for biliary atresia and may serve as a therapeutic target.

INTRODUCTION

Diagnostic accuracy and timeliness are critical for therapeutic interventions that target pathogenesis of diseases. When diseases are caused by well-defined sequence variants in gene or gene groups, the use of mutation screening tests simplifies diagnostic algorithms and allows for the personalization of treatments (1, 2). Unfortunately, the opportunities for molecular diagnosis or targeted treatments are limited in diseases of multifactorial etiology and complex pathogenesis (3, 4). Biliary atresia (BA) is such a disease prototype, with increasing evidence for viruses, environmental toxins, and susceptibility genes as etiologic agents, as well as a dysregulated immune response that disrupts the epithelial lining and limits tissue repair (58). This rare disease results from a rapidly progressive inflammatory and fibrosing injury to extrahepatic bile ducts (EHBDs), which interrupts the flow of bile and produces severe liver injury in otherwise healthy infants. With an onset of pathological jaundice limited to a highly reproducible window in the first 3 to 4 months of life of affected infants, early diagnosis and surgical intervention are critical for the restoration of bile flow and improvement in short-term outcome (7, 9, 10). However, neonatal jaundice is a common sign for several clinical syndromes. Clinical algorithms have been proposed to differentiate BA from other causes of neonatal cholestasis (7, 11, 12), but they are imprecise and have the potential to delay the diagnosis and treatment (12).

Previous studies analyzing the serum and plasma by metabolomics, enzyme-linked immunosorbent assays (ELISAs), and microRNA quantification have yielded potential biomarkers of BA (1315). However, the use of small cohorts and the lack of prospective validation in these studies limit the application of the findings in diagnosis or screening. Although circulating cytokines [for example, interleukin-2 (IL-2), IL-10, tumor necrosis factor–α (TNF-α), and IL-8] have been reported to play regulatory roles in pathogenesis of BA, they have not been shown to reliably differentiate BA from other cholestatic syndromes (16, 17). Here, we report the proteomic analysis of serum samples at the time of diagnosis of BA, which uncovered high circulating concentrations of matrix metalloproteinase-7 (MMP-7) above those of age-matched disease and healthy controls. After demonstrating the reproducibility of the findings in two validation cohorts, we explored the potential role of this marker in the pathogenesis of tissue injury. We found that MMP-7 is constitutively expressed by normal cholangiocytes, increases in the serum upon biliary injury, and modulates the clinical phenotype in an experimental model of BA.

RESULTS

Serum proteomics of BA in a discovery cohort

To search for biomarkers of BA, we applied the slow off-rate modified aptamer scan (SOMAscan, SomaLogic Inc.) to 70 serum samples from a discovery cohort of infants at the time of diagnosis of BA (at Kasai operation, n = 35) and to age-matched infants with neonatal intrahepatic cholestasis (IHC; n = 35; Fig. 1), as described previously (18). Infants were enrolled into a prospective study of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)–funded Childhood Liver Disease Research Network (ChiLDReN; www.childrennetwork.org; ClinicalTrials.gov identifier: NCT00061828). All groups were age-matched and had similar levels of hepatocellular injury and cholestasis (Table 1), except for a higher concentration of γ-glutamyltranspeptidase (GGT) in infants with BA (P < 0.0001). Of 1129 proteins (table S1), 76 were significantly overexpressed or underexpressed in BA compared to IHC [false discovery rate (FDR)–adjusted P < 0.05; table S2), with the top nine differentially expressed proteins shown in Fig. 2.

Fig. 1. Study design.

A total of 175 BA subjects and 70 subjects with IHC were randomized into the discovery cohort (BA = 35 and IHC = 35), validation cohort #1 (BA = 35 and IHC = 35), and validation cohort #2 (BA = 105). Serum proteins were measured by high-throughput proteomic assay SOMAscan) separately for individual cohorts. MMP-7, matrix metalloproteinase-7; ENPP7, ectonucleotide pyrophosphatase/phosphodiesterase family member 7.

Table 1. Clinical and biochemical characteristics of subjects.

Data are presented as the means ± SD. Mann-Whitney test was used. F, female; M, male; GGT, γ-glutamyltranspeptidase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; TB, total bilirubin.

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Fig. 2. Top nine proteins differentially expressed between BA and IHC.

Data from the discovery cohort categorized into BA (n = 35), IHC (n = 35), and age-matched normal controls (NCs; n = 9). Dots represent the log of relative fluorescent unit (RFU) for individual serum proteins. Bars and whiskers represent median and interquartile range, respectively. Q values from analysis of variance (ANOVA) with multiple hypothesis correction (Benjamini-Hochberg procedure). IGFBP-1, insulin-like growth factor-binding protein 1; ADAM9, disintegrin and metalloproteinase domain-containing protein 9; GCP-2, C-X-C motif chemokine 6; PIGR, polymeric immunoglobulin receptor; SARP-2, secreted frizzled-related protein 1.

To explore the potential value of these proteins as biomarkers of disease, we used multivariable logistic regression analyses and found that the combination of MMP-7 and ENPP7 distinguished BA from IHC with area under the receiver operating characteristic (ROC) curve (AUC) of 0.99 [95% confidence interval (CI), 0.97 to 1.00; Fig. 3, A and B]. Between the two proteins, MMP-7 alone showed excellent diagnostic features, with an AUC of 0.97 (95% CI, 0.93 to 1.00) and a sensitivity of 97% and specificity of 91% at a cutoff probability of 0.49. Taking into account the relatively low AUC of 0.78 for ENPP7 and the previous reports that serum GGT is generally higher in infants with BA (19, 20), we next examined how the combination of serum MMP-7 and GGT (available from the biochemical studies; n = 29 for BA and 31 for IHC; Fig. 3C) would compare to MMP-7 + ENPP7. Although GGT alone generated an AUC of 0.9 (95% CI, 0.83 to 0.97; Fig. 3, B and C), the AUC for MMP-7 + GGT at 0.98 (95% CI, 0.94 to 1.00) was just under the AUC for MMP-7 + ENPP7 at 0.99 (95% CI, 0.97 to 1.00). Collectively, the data on this discovery cohort were preliminary evidence that the combination of MMP-7 with ENPP7 or GGT had a potentially high diagnostic accuracy for infants with BA.

Fig. 3. Predictive features of MMP-7, ENPP7, and GGT for BA.

(A) ROC curves for serum MMP-7 and/or ENPP7 in distinguishing BA from IHC in multivariable logistic regression analyses and dot plots of the proteins in the discovery cohort. Data are shown as log of RFU; central horizontal lines and whiskers represent median and interquartile range. (B) Prediction models for MMP-7, ENPP7, and GGT. Sensitivity, specificity, and positive and negative predictive values were calculated at selected optimal cutoff. (C) Serum GGT concentrations in subjects with BA and IHC; normal range is 5 to 59 U/liter. The right panel shows ROC curves for MMP-7 and/or GGT. Q values from ANOVA with multiple hypothesis correction (Benjamini-Hochberg procedure) for (A) and P value from Mann-Whitney test for (C). NPV, negative predictive value; PPV, positive predictive value.

Validation of MMP-7 as a biomarker of BA

To test the reproducibility of the high accuracy for MMP-7, ENPP7, and GGT alone or in combination in BA, we performed the same SOMAscan assay in two other independent cohorts separately (Fig. 1). The first cohort consisted of the same number of age-matched infants with BA or IHC. To evaluate assay reproducibility, we included six samples from the discovery cohort to serve as bridging samples and found a high correlation (r = 0.9958; 95% CI, 0.9956 to 0.9960) between the two assays. Using the same analytical approach as in the discovery cohort, the different combinations of the serum biomarker and GGT produced lower AUCs than in the discovery cohort, except for a persistently high AUC of 0.94 for MMP-7 + GGT (95% CI, 0.88 to 1.00; Fig. 4A). In the second validation cohort, we performed a SOMAscan assay in 105 age-matched infants with BA and used the MMP-7 abundance signals and the serum GGT values in a discriminatory model for BA. Using the cutoff probability from the discovery cohort to assess the probability of a true positive and a false negative, the MMP-7 + GGT model showed a sensitivity of 96% to predict BA (table S3).

Fig. 4. MMP-7 biomarker validation, ROC curves according to serum GGT concentrations, and serum MMP-7 concentration.

(A) ROC curves in discovery and validation cohort #1 for MMP-7, ENPP7, and GGT. (B) ROC curves (95% CI) for subjects with GGT 100 to 300 U/liter (n = 10 for BA and n = 25 for IHC) and >300 U/liter (n = 50 for BA and n = 13 for IHC). Groups were formed with a combination of subjects with BA and IHC in the discovery cohort and validation cohort #1 based on the availability of GGT. (C) Bar graph of serum MMP-7 in randomly selected samples from patients with BA (n = 10), IHC (n = 10), and NCs (n = 4) measured by antibody-based ELISA compared with (D) the values measured by SOMAscan. Data are presented as median and interquartile range.

On the basis of the reproducibility of the findings in the validation cohorts, we next examined whether MMP-7 would aid in solving the clinical conundrum of differentiating BA from syndromes of IHC with high serum GGT. For this analysis, we hypothesized that serum MMP-7 maintains high discriminatory features for BA in subjects with serum GGT > 300 U/liter, a concentration that has been reported as more frequent in BA (19, 20). For this analysis, we combined all subjects in the discovery and validation cohort #1 (GGT concentrations available for 122 subjects), which were classified into groups as follows: (i) GGT <100 U/liter (n = 0 for BA and n = 24 for IHC), (ii) GGT = 100 to 300 U/liter (n = 10 for BA and n = 25 for IHC), and (iii) GGT > 300 U/liter (n = 50 for BA and n = 13 for IHC). The analysis for group (i) was not pursued because it contained no infant with BA. For groups (ii) and (iii), the AUCs for MMP-7 (0.92 and 0.88, respectively), MMP-7 + ENPP7 (0.91 and 0.93, respectively), and MMP-7 + GGT (0.93 and 0.88, respectively) were higher than for GGT (0.63 and 0.73, respectively) (Fig. 4B).

Examining the potential use of serum MMP-7 to diagnose infants with BA, we calculated the “number needed to misdiagnose” (NNM) as a means for estimating the number of patients assigned with the correct diagnosis of BA before a misdiagnosis occurs (21). The NNM for MMP-7 was 13.4 (meaning that 1 of 13.4 patients is misdiagnosed) and for MMP-7 + ENPP7 was 16.67, which were superior to the NNM of 5.4 for GGT (1 of 5.4 is misdiagnosed). Notably, the combination of MMP-7 + GGT had the best NNM at 19.1. Overall, this estimation emphasized the benefit of serum MMP-7 (alone or in combination) over GGT as a diagnostic aid for BA.

Validation by an antibody-based assay

To determine whether the high concentration of MMP-7 could be validated using a different technology, we obtained serum aliquots from 10 infants in the discovery cohort and determined the MMP-7 concentration using an antibody-based ELISA. The direct measurement of MMP-7 was high for BA and had no overlap with IHC (Fig. 4C). A comparative plot using SOMAscan data for the same subjects showed an even greater separation between the cohorts (Fig. 4D). Together, these data confirmed the high accuracy of MMP-7 + GGT for the diagnosis of BA, with validation in two independent cohorts and demonstration of technical reproducibility using an antibody-based assay.

Expression of MMP-7 in EHBDs and cholestatic syndromes

Searching for the biological basis of the increased serum concentrations of MMP-7 in BA, we first stained paraffin-embedded sections of the human liver (normal segment from liver tumor resection), gallbladder (normal histology after cholecystectomy of adult subject), and EHBDs (from a stillborn neonate). We focused on these tissues because of the predominant expression of MMP-7 in the liver and biliary system reported in the Human Protein Atlas (www.proteinatlas.org/ENSG00000137673-MMP7/tissue). Our immunostaining showed minimal or no expression of MMP-7 in parenchymal or nonparenchymal cells of the normal liver. By contrast, the protein was specifically expressed in the cytoplasm of cholangiocytes along the epithelium of the gallbladder, EHBD, and the surrounding peribiliary glands (Fig. 5A).

Fig. 5. MMP-7 expression in human liver, EHBD, and gallbladder.

(A) Immunohistochemical staining of normal human liver shows minimal or no expression of MMP-7 in hepatocytes and IHBDs but intense expression in the biliary epithelium of gallbladder (GB) and EHBD. Scale bars, 50 μm (in liver, GB, and EHBD) and 20 μm (in IHBD). (B) Variable intensity of expression (0 to 3+) of MMP-7 in the cytoplasm of intrahepatic biliary epithelium of BA subjects. (C) Livers of patients with α1-antitrypsin deficiency had lower MMP-7 expression in the IHBDs. Scale bar, 20 μm for both (B) and (C). (D) Percentage of livers showing different intensities of MMP-7 immunostaining in patients with BA and α1-antitrypsin (A1AT) deficiency. (E) Hepatic mRNA expression of MMP-7 for BA (n = 64), IHC (n = 14), and NC (n = 7). Expression is normalized to NCs and presented as fold change. P values are from Kruskal-Wallis test.

In liver biopsies obtained at the time of diagnosis of BA, MMP-7 was detected in cholangiocytes of intrahepatic bile ducts (IHBDs) in most samples, albeit at variable levels, and in a few hematopoietic cells within the portal tracts (n = 12; Fig. 5B). A lighter expression pattern was detected in liver biopsies of infants with α1-antitrypsin deficiency (n = 6; Fig. 5C) and idiopathic neonatal cholestasis (n = 2), which may account for the increased serum concentration in IHC above healthy controls (Fig. 5D). To determine MMP-7 expression at the transcriptional level, we extracted liver mRNA data from the genome-wide expression data sets for subjects with BA (n = 64), IHC (n = 14), and normal controls (n = 7), which we published previously and submitted to the Gene Expression Omnibus (GSE46995) (17). Consistent with histological findings, livers from BA subjects had higher MMP-7 mRNA expression compared to IHC (fold change relative to normal controls, 9.98 ± 7.65 versus 2.54 ± 1.73; P < 0.0001; Fig. 5E). The mRNA expression for GGT in these biopsies was not significantly different between the groups, revealing a discrepancy at the hepatic transcriptional level when compared to the differences in serum GGT (fig. S1). Together, these data defined a primary localization of MMP-7 in cholangiocytes of EHBDs, with minimal or no expression in intrahepatic cholangiocytes of normal livers and detectable expression in intrahepatic cholangiocytes of diseased livers (but lower than the level seen in EHBDs). At the whole tissue level, the total liver MMP-7 mRNA was still higher in BA when compared to diseased controls.

Lack of correlation between MMP-7 and fibrosis

To explore the biological properties of MMP-7 in determining the phenotype of extrahepatic cholangiopathies, we first investigated the relationship between MMP-7 expression and tissue fibrosis. On the basis of the well-described role of metalloproteinases in the turnover and degradation of extracellular matrix and on the previous reports linking MMP-7 to elastin, E-cadherin, and syndecan-1 (2224), we hypothesized that MMP-7 expression would correlate with the severity of liver fibrosis. To test this hypothesis, we analyzed the relationship between serum MMP-7 and histological fibrosis using the Scheuer fibrosis staging 0 to 4, as described previously (25). For these experiments, the fibrosis staging was performed independently by the pathologists of the ChiLDReN consortium in liver biopsy samples from the same subjects with BA whose sera were analyzed by the SOMAscan assay. Serum MMP-7 correlated poorly with liver fibrosis staging at the time of diagnosis (Spearman r = 0.33; 95% CI, 0.17 to 0.48; P < 0.0001; fig. S2A). Similarly, ROC analysis showed that serum MMP-7 was a poor predictor of hepatic fibrosis (AUC, 0.69; 95% CI, 0.59 to 0.78; fig. S2B). To investigate whether the expression of MMP-7 correlated with fibrosis within the same tissue, we compared the surface area of hepatic fibrosis detected by trichrome staining and of the area immunostained by MMP-7 (fig. S2C). Again, MMP-7 expression in the liver had very low correlation with hepatic fibrosis (Spearman r = 0.25; P = 0.52; fig. S2D). Collectively, these data did not support a functional role of MMP-7 in modulating the fibrosing phenotype at the time of diagnosis of BA.

Increase of MMP-7 upon experimental biliary injury

Without evidence that MMP-7 is linked to hepatic fibrosis in BA, we next explored whether the protein is involved in the inflammatory mechanisms that regulate epithelial injury. On the basis of the proposed role of MMP-7 in activating the innate immune response to a tissue injury (26), we first determined whether serum concentrations of MMP-7 increase in neonatal mice after bile duct epithelial injury induced by rotavirus infection. In this model, the intraperitoneal administration of 1.5 × 106 fluorescent-forming units of rhesus rotavirus type-A (RRV) into BALB/c mice within 24 hours of birth injures the epithelial lining of EHBD, followed by an inflammatory obstruction of the lumen and atresia (27, 28). As expected, RRV infection resulted in obstruction of EHBD, jaundice, growth retardation, and acholic stools; universal mortality occurred by day 15 of viral challenge (29). Analyzing serum samples collected 7 days after RRV injection (time of the peak of epithelial injury), the concentration of MMP-7 increased significantly above that of age-matched, saline-injected controls (2.01 ± 0.39 versus 0.96 ± 0.14 ng/ml; n = 3 to 4 per group; P = 0.03; Fig. 6A). In the liver, real-time polymerase chain reaction (RT-PCR) revealed that Mmp-7 expression significantly increased at 14 days after RRV injection (P < 0.0001; Fig. 6B), and in EHBD, Mmp-7 increased by 20- and 6-fold higher than age-matched controls at 7 and 14 days, respectively, after RRV injection (P < 0.0001; Fig. 6C). This pattern of mRNA expression differed from Ggt1, which only increased in the liver 3 days after RRV (fig. S3, A and B).

Fig. 6. MMP-7 expression in experimental BA.

(A) Serum MMP-7 measured in mice 7 days after RRV or saline injection (n = 3 to 4 per group). Means ± SEM, P values are from unpaired t test. (B and C) Mmp-7 mRNA expression in the livers (B) and EHBDs (C) at 3, 7, and 14 days after RRV or saline injection (n = 3 to 4 per group and per time point). Values are normalized to Gapdh and are expressed as means ± SEM; P values were for the comparison between two groups at each time point (ANOVA). (D) Liver immunohistochemistry detects MMP-7 in extramedullary hematopoietic cells (arrowheads) in saline-injected mice predominantly at day 3. Hepatic hematopoietic cells decrease after RRV, with MMP-7 being detected in inflammatory cells (arrows) infiltrating the portal tracts at days 7 and 14 after RRV injection. There was no staining of IHBD epithelium. (E) MMP-7 is detected in the duct epithelium in RRV and saline injected groups. At days 7 and 14 after RRV injection, MMP-7 is also detected in inflammatory cells and injured epithelium. Asterisks depict the duct lumen (or lack thereof); scale bars, 20 μm [for (D) and (E)].

To examine whether the cellular source of MMP-7 expression in neonatal mice was similar to the pattern in humans, we immunostained livers and EHBD at 3, 7, and 14 days of life of saline-injected mice (serving as controls) and after RRV injection. In control mice, the hepatic expression of MMP-7 was detected in extramedullary hematopoietic cells and restricted to the first 3 days of life, whereas the pattern in EHBDs was predominantly localized to cholangiocytes lining the entire length of the duct epithelium, cholangiocytes of neighboring peribiliary glands, and in few subepithelial cells at all time points (Fig. 6, D and E). After RRV infection, MMP-7 was detected in inflammatory cells infiltrating the periductal space of portal tracts at 7 and 14 days (Fig. 6D), with a broader staining pattern in epithelial cells and subepithelial compartment of EHBD (Fig. 6E). Together, the immunostaining pattern revealed a striking similarity with the findings in human tissues, in which MMP-7 is expressed predominantly by cholangiocytes of EHBDs, with a release to the serum triggered by the epithelial injury. These findings raised the possibility that MMP-7 may be involved in the regulation of the phenotype of experimental BA.

Suppression of experimental BA by batimastat and MMP-7 antibody

To directly test whether MMP-7 plays a mechanistic role in pathogenesis of biliary injury, we injected the MMP inhibitors batimastat (with activity against MMP-7) or GM6001 (without activity against MMP-7) intraperitoneally on days 1, 3, 5, and 7 after RRV injection; the vehicle [5% dimethyl sulfoxide (DMSO), 28.5% propylene glycol, 5% Tween 80, and 62% of 0.9% NaCl] was injected into a separate group of RRV-infected mice serving as controls. First, we evaluated whether other MMPs were up-regulated in the experimental model at the time of bile duct obstruction and onset of symptoms (7 days after RRV challenge). We found minimal or no change in the mRNA expression of Mmp-2, Mmp-3, Mmp-8, Mmp-9, Mmp-10, Mmp-12, Mmp-13, and Mmp-14 in the liver and EHBD, with only Mmp-7 increasing in the liver by 2-fold and in EHBD by 89-fold above controls (Fig. 7A). The RRV-infected experimental and control mice injected with batimastat, GM6001, or vehicle were sacrificed at 12 to 14 days after RRV injection for tissue analyses. In the liver, the histological features of portal inflammation and hepatocellular necrosis were mild in batimastat-treated mice and moderate or severe in mice treated with vehicle or GM6001 (Fig. 7B). The beneficial effects of batimastat were even more pronounced in EHBDs, where it prevented epithelial injury and duct obstruction in 86% of neonatal mice, whereas all EHBDs were obstructed in mice injected with GM6001 or vehicle (Fig. 7, C and D). Livers of mice treated with batimastat had suppressed mRNA expression for Tnfa, Cxcl1, Cxcl2, Cxcl5, and Cxcl9 14 days after RRV infection, without changes in Cxcl10, Cxcl11, Ifng, or IL12p40 (Fig. 7E). These findings suggested that the potential mechanisms of tissue injury used by MMP-7 are directly or indirectly linked to TNF-α and IL-8 circuits (29).

Fig. 7. Suppression of tissue injury, inflammation, and cytokine expression by batimastat and MMP-7 antibody in experimental BA.

Neonatal BALB/c mice were injected with RRV in the first day of life and then were injected with batimastat (RRV + batimastat; n = 22), GM6001 (non–MMP-7, RRV + GM6001; n = 17), anti–MMP-7 antibody (RRV + MMP-7 Ab; n = 12), or vehicle in the control group (RRV + vehicle; n = 14). (A) mRNA expression of tissue Mmps in the liver and bile duct at 3 and 7 days after viral injection. (B) Representative liver sections showing variable degrees of portal inflammation and hepatic necrosis in different groups. (C) Representative sections of EHBDs in each group. Asterisks depict the duct lumen (or lack thereof). Scale bars, 100 μm for (B) and 20 μm for (C). (D) Graphs depict the frequency of mice with liver injury, according to the degree of inflammation and hepatocellular necrosis, and frequency of mice with obstructed lumen. (E) Hepatic mRNA expression for cytokines/chemokines 12 days after RRV injection and treatment with batimastat or vehicle (as controls). P values are from ANOVA for (A), chi-square test for (D), and unpaired t test for (E).

To more rigorously examine the impact of inhibition of MMP-7 on the development of the obstructive phenotype in experimental BA, we injected neutralizing anti–MMP-7 antibodies (AF-907, Bio-Techne) intraperitoneally daily to 12 newborn mice at days 1 to 5 after RRV inoculation; control mice received goat serum. All mice were sacrificed 12 days later. Histologically, the liver injury varied from mild to moderate, with a notable prevention of the duct obstruction, as shown by patency of EHBDs in 82% of the mice similarly to the phenotype observed after administration of batimastat (Fig. 7, B to D), whereas all control mice receiving goat serum had full obstruction of EHBDs.

DISCUSSION

By applying large-scale quantitative proteomics to sera of infants with BA, we found high circulating concentrations of soluble proteins at the time of diagnosis of BA. Chief among them was MMP-7, whose expression distinguished the disease from other causes of IHC. The prospective validation of these findings in two independent cohorts pointed to the value of MMP-7 as a biomarker of BA, with an even greater discriminatory value when linked to the serum concentrations of GGT. Searching for the biological basis of high circulating MMP-7, in situ immunostaining localized MMP-7 expression in cholangiocytes along the epithelium of EHBDs and gallbladder of normal subjects, and to a lower extent, in intrahepatic cholangiocytes of diseased livers. A similar expression pattern was present in neonatal mice, in which modeling of the disease demonstrated a release of MMP-7 into the circulation upon epithelial injury. Notably, pharmacologic inhibition by batimastat and antibody neutralization of MMP-7 suppressed the experimental BA phenotype, with lower expression of Tnfa, Cxcl9, and the three murine orthologs of IL8 and decreased tissue injury. Together, these findings support a role for serum MMP-7 as a biomarker of BA and a distinct cellular expression in the extrahepatic epithelium that suggested a direct implication in pathogenesis of disease.

The discovery of minimally invasive biomarkers highly predictive of BA is crucial for the timely diagnosis and stratification of care. Despite the well-recognized value of young age as a key factor associated with the best surgical outcome, the overlapping clinical and biochemical features shared between BA and other causes of neonatal cholestasis create a formidable diagnostic challenge that leads to a delay in surgery. Here, the finding that the quantification of a single protein biomarker has an accuracy of 94.3% for BA has the potential to greatly simplify diagnostic approaches, especially when the findings are validated in two separate cohorts and by a different assay/technology.

Among the published biomarkers of BA, serum GGT concentrations have the highest sensitivity and specificity of 40 and 98%, respectively (19), which may reach up to 83 and 82% in infants aged 61 to 90 days (20). The analysis of the behavior of GGT in our cohorts showed higher concentrations in BA, with 83% sensitivity and 81% specificity to predict the disease. When we coupled serum MMP-7 with GGT in a composite model, we obtained higher sensitivity and specificity of 97 and 94%, respectively, at optimal cutoff, which provided positive and negative predictive values of 85 and 99%, respectively, if one considers the prevalence of BA of 25.9% among infants with conjugated hyperbilirubinemia (30). Other candidate biomarkers previously reported include miR-140-3p (31), miR-200b/429 (14), miR-4429, and miR-4689 (32) but with lower sensitivity (66 to 83%) and specificity (79 to 83%) and no prospective validation. In a separate study, the use of two-dimensional gel electrophoresis with tandem mass spectrometry found a combination of 11 proteins as biomarkers; the small cohort size and relatively large number of protein analytes selected in this study limit the interpretation of the results and their potential use in clinical practice (33). Together, these data suggest a superior value for MMP-7 + GGT among circulating biomarkers of BA. Their predictive features may extend beyond the published predictive value of liver histology in BA (25, 34). In fact, assuming the existence of some degree of variability among pathologists in interpreting histological features of biliary obstruction (25), the noninvasive, non–operator-dependent features suggest that the measurement of serum MMP-7 + GGT early in diagnostic algorithms may substantially facilitate the evaluation of cholestatic infants and establish the diagnosis of BA.

The singular emergence of MMP-7 from a comprehensive proteomic analysis points to a potential link to pathogenic mechanism(s) of disease. Previous studies have reported MMP-7 as a diagnostic or prognostic marker for various malignancies, including cholangiocarcinoma, pancreatic carcinoma, and gastric cancer, as well as fibrosis of lung and liver (3539). Specifically relevant to our findings, MMP-7 was previously reported as increased in serum and liver samples from subjects with BA after surgical treatment (40, 41), correlating with the severity of fibrosis regardless of the degree of jaundice clearance (41). However, testing this possibility, we found no or weak correlation between the expression of MMP-7 in the serum or in the liver and the degree of hepatic fibrosis at the time of diagnosis. It remains possible that the concentrations of serum MMP-7 may change after surgical treatment and be a useful marker of progression of tissue fibrosis.

The close relationship between MMP-7 and BA suggests a potential role for MMP-7 in pathogenesis of disease. Its predominant expression in normal extrahepatic cholangiocytes and the variable detection in intrahepatic ducts of diseased livers (from patients with BA and IHC) link its increase in the serum to a response to tissue injury. Making use of the mouse model of experimental BA, we directly demonstrated this possibility by the timely rise of serum MMP-7 after rotavirus injury of EHBDs. In published reports by other investigators, the release of MMP-7 after injury of the lung epithelium was shown to promote shedding of syndecan-1 and CXCL-8 and create a pericellular chemokine gradient that controls the influx and activation of neutrophils at the injury site (26). Furthermore, MMP-7 may modulate tissue repair by cleaving E-cadherin and disrupting adherens junctions to facilitate cell migration and inflammation by activation of FAS ligand and latent TNF-α (26, 42). Therefore, we performed proof-of-principle studies for investigating the role of MMP-7 in pathogenesis of biliary injury by using antibody neutralization or inhibition by batimastat. Both agents prevented the obstruction of EHBDs in >80% of neonatal mice infected with RRV and decreased the expression of IL-8 orthologs and TNF-α in mice treated with batimastat.

We recognize that acquisition and analysis of samples and data in a prospective fashion are important to determine the diagnostic utility of MMP-7. To overcome the intrinsic challenge of designing such a study in a disease of very low incidence, such as BA, we used blood samples and clinical data collected prospectively from subjects over several years and then simulated a prospective study design by randomly assigning the samples into the discovery and validation cohorts before any assay or analysis. Next, we performed the SOMAscan and analyzed the data in the discovery cohort. Only then, we sequentially validated the findings in the two test cohorts, thus strengthening the evidence for MMP-7 as a biomarker of disease. Another limitation is that our study design did not include multiple subcohorts of infants with specific syndromes of neonatal cholestasis. This limitation was largely due to the rare nature of these diseases and to our objective of building a disease cohort that will more closely replicate the most common clinical scenario, which includes idiopathic neonatal cholestasis (most of which has spontaneous recovery) and a few patients with specific syndromes, such as Alagille syndrome and deficiencies of canalicular transporters, that are not readily distinguishable clinically. With these limitations notwithstanding, we report a distinct increase in serum MMP-7 at the time of diagnosis of BA, with concentrations above those in age-matched diseased and normal controls and high predictive features as a biomarker of disease. Combining these findings with the rise of serum MMP-7 after experimental biliary injury and the preliminary evidence that it modulates tissue injury and inflammation, we propose that MMP-7 has a role in pathogenesis of disease. Clinically, the implications of our findings relate to the potential incorporation of serum MMP-7 alone or in combination with GGT in diagnostic algorithms in the clinical setting. It remains to be investigated whether the longitudinal measurement of serum MMP-7 may also be used to monitor progression of liver injury and fibrosis.

MATERIALS AND METHODS

Study design

Human subjects were infants with cholestasis enrolled into the Prospective Database of Infants with Cholestasis (PROBE study; ClinicalTrials.gov identifier: NCT00061828) of the NIDDK-funded ChiLDReN (www.childrennetwork.org). PROBE was designed to acquire longitudinal, prospective clinical, and laboratory data in a standardized fashion at defined time points. Serum samples from BA or IHC subjects were randomly assigned into the discovery or validation cohort #1 (with similar numbers of subjects) before any assay or analysis. A second validation cohort consisted of 105 samples from BA subjects. SOMAscan proteomics was performed in individual cohorts sequentially to enable the discovery of candidate protein biomarkers, followed by validation in the two cohorts (Fig. 1).

For mouse studies, newborn mice were randomly assigned into an “infection group,” injected with 1.5 × 106 fluorescent-forming units of RRV or into a “control group,” injected with an equal volume as 0.9% NaCl (saline) within 24 hours after birth to induce experimental biliary injury and obstruction according to protocols published previously (29). Mice were monitored daily, and subgroups of mice were randomly selected (independently of the presence or absence of jaundice or other phenotypic feature) and sacrificed at days 3, 7, and 14 for serum and tissue examination. Additional groups of mice were injected intraperitoneally with 30 μg/day of batimastat (BB-94, ApexBio) or 3 μg/day of GM6001 (ApexBio) daily at 1, 3, 5, and 7 days after RRV injection; the vehicle (5% DMSO, 28.5% propylene glycol, 5% Tween 80, and 62% of 0.9% NaCl) was used in the control group. A separate group of neonatal mice received 10 μg of goat-derived, anti-human MMP-7 antibody (AF-907, Bio-Techne) intraperitoneally at days 1 to 5 after RRV infection; controls included a similar volume of goat serum.

Human samples

Sera were collected at the time of diagnosis (initial evaluation before or at the time of Kasai portoenterostomy) from 175 BA subjects; 70 other subjects with IHC served as disease controls. The diagnosis of BA was confirmed by the finding of luminal obstruction of EHBD by histopathological examination. The diagnoses of IHC included idiopathic neonatal hepatitis (n = 1), Alagille syndrome (n = 2), progressive familial IHC (n = 1), cytomegalovirus hepatitis (n = 1), neonatal sclerosing cholangitis (n = 1), mitochondrial DNA depletion syndrome (n = 1), endocrinopathy (n = 1), and cholestasis of unknown etiology (n = 62). Serum samples were also obtained from nine age-matched subjects (without history of previous liver disease) to serve as normal controls. Tissue sections for immunostaining were obtained from deidentified paraffin-embedded liver tissues archived in the biobank repository of Cincinnati Children’s Hospital Medical Center (CCHMC); the diagnosis followed the same criteria used for subjects enrolled in ChiLDReN. All samples were obtained after informed consent from patients’ parents/legal guardians. The study protocols were approved by the human research review boards of all participating institutions.

Mice and histological analyses

Newborn mice were of the BALB/c strain and were housed in a temperature-controlled, 12-hour light cycle, and specific pathogen-free environment. Histological analyses of tissues stained with hematoxylin and eosin were performed by an investigator unaware of the experimental or control origin of samples. EHBDs were classified as obstructed or patent, and liver histology was scored according to portal inflammation and area of lobular necrosis as follows: 1 - mild portal inflammation without area of necrosis (mild), 2 - mild to moderate portal inflammation with patchy necrosis (moderate), and 3 - moderate to severe portal inflammation with large area of necrosis (severe). The Animal Care and Use Committee at CCHMC approved all experiments involving laboratory animals.

Proteomic assay and MMP-7 concentration

Serum samples were subjected to the SOMAscan protein analysis platform (SomaLogic Inc.). The assay measures 1129 proteins simultaneously using aptamer-based technology; detailed assay protocols and specificity have been described elsewhere (18). To determine the interassay reproducibility, we included six samples in every run to serve as bridging samples.

MMP-7 concentrations were determined by ELISA using MILLIPLEX Multiplex kits (Millipore) according to the manufacturer’s protocol. Briefly, 25 μl of sample in duplicate was incubated with 25 μl of antibody-coated beads in a 96-well multiscreen filter plate overnight at 4°C on a plate shaker. Plates were then washed twice on a vacuum apparatus, followed by the addition of 25 μl of secondary antibody, and incubation at room temperature for 1 hour with gentle shaking. To the plate, 25 μl of streptavidin–R-phycoerythrin were added and incubated for 30 min and then washed and read using the Bio-Plex (Bio-Rad). Concentrations were calculated from standard curves using recombinant proteins and expressed in nanograms per milliliter.

Histology, immunohistochemistry, and automated image analysis

The correlation of liver fibrosis with serum MMP-7 used histological grade scores generated by ChiLDReN pathologists for liver biopsy samples from the same subjects with BA whose sera were analyzed by the SOMAscan assay. Immunohistochemistry was performed on 5-μm paraffin-embedded tissue sections from human subjects and experimental mice, as described previously (43), using 1:50 polyclonal MMP-7 antibody (Cloud-Clone Corp.) in blocking serum overnight at 4°C. Biotinylated anti-rabbit antibody (Vector Laboratories) was used as secondary antibody followed by avidin/biotin detection (VECTASTAIN ABC reagent PK-4001; Vector Laboratories) and the 3,3′-diaminobenzidine (DAB) substrate (Vector kit, SK-4100; Vector Laboratories). The same protocol was used in human and mouse tissues. An additional liver biopsy slide from each BA subject was subjected to Masson’s trichrome staining. For image analyses, entire sections were scanned and subdivided to depict the portal tracts using Olympus BX51 microscope (Olympus America Inc.) and cellSens Dimension digital imaging software (version 1.8.1, Olympus Corporation). Automated quantification of MMP-7 expression was performed on scanned images with NIS Nikon Elements software using red-green-blue (RGB) general analysis. Positive expression was defined as DAB-positive (DAB+) brown area. Portal tracts were selected and enumerated as regions of interest. RGB general analysis was used in hue, saturation, and intensity modes to identify DAB+ areas. All subdivided regions were averaged to report surface area of DAB+ per portal tract in each sample. For Masson’s trichrome staining, positive collagen was defined by blue areas with use of hue, saturation, and intensity modes. The quantification was reported as surface area of collagen (blue-stained)/portal tract.

Microarray analysis

We obtained data on MMP-7 expression from the genome-wide expression data sets of human liver samples of BA, IHC, and healthy controls (as above) reported by us previously and submitted to the Gene Expression Omnibus (data accessible at NCBI GEO database, accession GSE46995) (17). The data sets were extracted as Affymetrix CEL files and analyzed with GeneSpring software (Agilent Technologies).

Quantitative PCR

Total RNA was isolated from the livers and EHBDs of RRV- or saline-injected BALB/c mice with or without batimastat or vehicle injection using the RNeasy Mini Kit, according to the manufacturer’s protocol (Qiagen Inc.); RNA integrity was verified by agarose gel electrophoresis as described previously (29). All samples were then reverse-transcribed to cDNA using the High-Capacity RNA-to-cDNA kit (Applied Biosystems) and used for RT-PCR on a Stratagene Mx3000P thermocycler (Agilent Technologies Inc.) to detect relative concentrations of individual mRNAs. PCR amplifications were performed with specific primers of Mmp-7, Ggt1, Cxcl1, Cxcl2, Cxcl5, Cxcl9, Cxcl10, Cxcl11, Tnfa, Ifng, and IL12p40 (table S4). mRNA expression of target genes was normalized to the endogenous reference Gapdh gene.

Statistical analysis

Without a priori data on the serum concentration of 1129 proteins in the first year of life, we did not estimate a sample size, and instead, built a discovery cohort with a convenience size of n = 35 each for BA and for IHC. Then, we developed a validation cohort #1 that matched the numbers for BA and IHC (n = 35 for each) and additional samples of BA only as a validation cohort #2 (n = 105), without inclusion of IHC, due to the lack of available samples. All protein results were log-transformed to accommodate the wide range of assayed proteins and skewness of their measurement values. Statistical analyses and graphic presentation were performed using SAS software, version 9.3. First, we chose the candidate proteins using Student’s t test with FDR correction (Benjamini-Hochberg procedure) in the discovery cohort; significance criteria were adjusted P (Q value) < 0.05 for BA versus IHC. Then, we applied multivariable stepwise logistic regression analysis to the list of candidate proteins to identify proteins that best discriminate BA from IHC. The diagnostic performance of the biomarkers was assessed by analyzing ROC curves and by calculating the AUC of each model. The sensitivity (true positive) and the specificity (true negative) were determined at optimal cutoff probability. Positive and negative predictive values were calculated using prevalence of BA among infants with cholestasis reported in the literature (30). Likewise, the ROC analyses were used to validate the diagnostic performance of each model in the validation cohort #1. In the second validation, because the cohort consisted of BA subjects only, we used the cutoff probability from the discovery cohort to assess the probability of true positive and false negative, followed by calculation of sensitivity. Other statistical analyses included ANOVA, Kruskal-Wallis test, and Spearman and Pearson correlations. Differences were considered statistically significant at P < 0.05. The NNM was calculated as described previously (21).

SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/9/417/eaan8462/DC1

Fig. S1. GGT mRNA expression.

Fig. S2. Relationship between MMP-7 and hepatic fibrosis at the time of diagnosis of BA.

Fig. S3. Murine Ggt1 mRNA expression.

Table S1. Protein results from SOMAscan (provided as an Excel file).

Table S2. List of 76 serum proteins differentially expressed in BA and IHC.

Table S3. Prediction of BA by MMP-7 in validation cohort #2.

Table S4. Primer sequences and PCR product sizes for mouse Mmp-7-, Ggt1-, Th1-, Tnfa-, and IL8-related genes.

REFERENCES AND NOTES

Acknowledgments: The studies were supported by Junior Co-Operative Society of CCHMC. Serum and liver tissues were obtained as an ancillary study of the NIDDK-funded ChiLDReN (DK-62497). We thank the pathologists of ChiLDRen for reviewing and scoring all histological specimens, the Data Coordinating Center for managing all studies and providing data and specimens, and the principal investigators and clinical research coordinators of individual ChiLDReN Centers for patient recruitment and acquisition of tissue and data. The contents of the article do not necessarily reflect the opinions or views of the NIDDK, ChiLDReN, or the ChiLDReN investigators. Funding: This study was supported by the NIH grants DK-64008, DK-62497, and DK-83781 to J.A.B. and by the Integrative Morphology and the Gene Analysis Cores of the Digestive Health Center (DK-78392). Author contributions: J.A.B., P.S., and C.L. designed the research work. P.S., C.L., L.Y., and S.G. performed the animal experiments. J.A.B., P.S., C.L., R.M., and K.E.B. analyzed the data. L.F., Y.Z., and C.L. performed the statistical analyses. C.L. and J.A.B. wrote and edited the manuscript. All contributing authors assisted in reviewing the manuscript. Competing interests: The authors declare that they have no competing interests.
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