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Markers of Tissue Repair and Cellular Aging Are Increased in the Liver Tissue of Patients With HIV Infection Regardless of Presence of HCV Coinfection

Markers of Tissue Repair and Cellular Aging Are Increased in the Liver Tissue of Patients With... Downloaded from https://academic.oup.com/ofid/article-abstract/5/7/ofy138/5035423 by Ed 'DeepDyve' Gillespie user on 16 October 2019 Open Forum Infectious Diseases BRIEF REPORT activation of hepatic stellate cells (HSCs) via the Toll-like recep- Markers of Tissue Repair and Cellular tor 4, cysteine-X-cysteine receptor 4, and chemokine motif Aging Are Increased in the Liver receptor 5 receptors [3, 4]. Tissue of Patients With HIV Infection Fibrosis is a wound-healing response to tissue (epithe- lial) injury. The regenerative responses of wound-healing dif- Regardless of Presence of HCV fer depending on the severity and chronicity of liver injury. Coinfection Although initially beneficial, the repair process can become 1,2 3 1,4 2 Susanna Naggie, Marzena Swiderska-Syn, Steve Choi, Sam Lusk, pathogenic if it continues aberrantly, leading to considerable 5 1 3,6 1 1 Audrey Lan, Guido Ferrari, Wing-Kin Syn, Cynthia D. Guy, and Anna Mae Diehl tissue remodeling and scar. The fetal morphogen hedgehog 1 2 Duke University School of Medicine, Durham, North Carolina; Duke Clinical Research 3 (Hh) signaling pathway has been implicated in multiple met- Institute, Durham, North Carolina; Medical University of South Carolina, Charleston, South 4 5 Carolina; Durham VA Medical Center, Durham, North Carolina; University of North Carolina abolic diseases, including nonalcoholic steatohepatitis, obesity, School of Medicine, Chapel Hill, North Carolina; Ralph H. Johnson VA Medical Center, and diabetes [5–7]. The Hh pathway has also been implicated Charleston, South Carolina in HCV mono-infection, which induces hepatic steatosis and insulin resistance [8], and now for the first time the Hh pathway Liver disease is a leading cause of HIV-related mortality. Hepatitis has been implicated in HIV-associated nephropathy [9]. C virus (HCV)–related fibrogenesis is accelerated in the setting of HIV coinfection, yet the mechanisms underlying this aggres- e Hh p Th athway, originally identified in Drosophilia , is a sive pathogenesis are unclear. We identified formalin-fixed paraf- highly conserved signaling pathway that orchestrates multiple fin-embedded liver tissue for HIV-infected patients, HCV-infected aspects of organogenesis and tissue remodeling [10]. Hh path- patients, HIV/HCV-coinfected patients, and controls at Duke way activation typically enhances the growth and viability of University Medical Center. De-identified sections were stained for Hh-responsive cells, whereas abrogating Hh signal transduction markers against the wound repair Hedgehog (Hh) pathway, res- usually triggers apoptosis, conferring a selective advantage for ident T-lymphocytes, and immune activation and cellular aging. cell types capable of responding to Hh [11]. Hh-ligands upreg- HIV infection was independently associated with Hh activation ulate expression of chemokines and receptors in immature and markers of immune dysregulation in the liver tissue. liver epithelial cells, thus facilitating hepatic recruitment and Keywords. b fi rogenesis; GLI; Hedgehog; hepatitis C virus; retention of particular types of immune cells [12]. Specifically, human immunodeficiency virus; patched; pathogenesis; Sonic activated NKT cells are recruited, and by avoiding apoptosis, Hedgehog; wound repair. they accumulate in liver tissue, in turn enhancing liver disease progression. Gli2, a Hh pathway transcription factor, is also reported to regulate transcription of TGFß-1, a key prob fi ro- Liver disease is a leading cause of HIV-related morbidity and genic cytokine implicated in HIV/HCV-related fibrogenesis mortality [1]. In the United States, chronic hepatitis C virus [13]. (HCV) infection is the leading cause of liver disease and related Using liver tissue specimens from HIV-infected patients, mortality in HIV infection [1]. HCV is a metabolically active HCV-infected patients, HIV/HCV-coinfected patients, and con- virus that, in the setting of HIV coinfection, results in acceler- trols, we assessed for markers of the Hh pathway, T-lymphocyte ated liver disease pathogenesis [2]. The mechanisms identified cellular subpopulations, cellular aging and senescence, and as potentially contributing to this more severe disease pathogen- b fi rogenesis. We proposed testing the hypothesis that Hh sig- esis include increased oxidative stress and downstream induc- naling is aberrantly activated in the liver tissue of HIV-infected tion of prob fi rotic pathways by cytokines including TGFß-1 and patients with HCV and that this increased Hh activity correlates with increased liver NK T-cell populations and increased mark- Received 13 March 2018; editorial decision 30 May 2018; accepted 8 June 2018; published ers of fibrosis. online June 11, 2018. Correspondence: S. Naggie, MD, MHS, Infectious Diseases Section, Duke Clinical Research Institute, 2400 Pratt Street, Durham, NC, 27705 (susanna.naggie@duke.edu). METHODS Open Forum Infectious Diseases © The Author(s) 2018. Published by Oxford University Press on behalf of Infectious Diseases Study Population Society of America. This is an Open Access article distributed under the terms of the Creative Formalin-fixed paraffin-embedded (FFPE) liver tissue sections Commons Attribution-NonCommercial-NoDerivs licence (http://creativecommons.org/licenses/ by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any were identified for HIV-infected patients, HCV-infected patients, medium, provided the original work is not altered or transformed in any way, and that the work HIV/HCV-coinfected patients, and de-identified controls from is properly cited. For commercial re-use, please contact journals.permissions@oup.com the Department of Pathology at Duke University Medical Center, DOI: 10.1093/ofid/ofy138 BRIEF REPORT • OFID • 1 Downloaded from https://academic.oup.com/ofid/article-abstract/5/7/ofy138/5035423 by Ed 'DeepDyve' Gillespie user on 16 October 2019 in accordance with National Institutes of Health (NIH) and models were built and adjusted for independent variables: sex, institutional guidelines for human subjects research. All HIV/ race, age, METAVIR fibrosis stage, METAVIR inflammation HCV-coinfected patients with available liver biopsy specimens score, steatosis, HIV infection, HCV infection, alanine ami- who did not have any exclusion criteria were included. HCV- notransferase (ALT), aspartate aminotransferase (AST), and monoinfected patients were then identified by matching to HIV/ diabetes. An interaction term was included to assess the effect HCV-coinfected patients by age (±5  years) at the time of liver of HIV/HCV coinfection when compared with monoinfection. biopsy, sex, and METAVIR stage (0–1 and 2–4) of liver fibrosis If the HIV/HCV coinfection interaction term is reported as on the biopsy. Due to the small number, all HIV-monoinfected significant, it suggests that HIV/HCV coinfection has a unique patients with available liver biopsy who did not have any exclu- impact on the staining of the marker of interest beyond that of sion criteria were included. Reasons provided on the requisition HIV or HCV monoinfection. Controls were not included in the form for liver biopsy for the HIV-monoinfected patients included regression models due to the lack of clinical data. Additional abnormal liver enzymes and steatosis noted on imaging (n = 4), multivariate linear regression models were used to determine ill-defined lesions on imaging (liver tissue was normal), abnor- if HIV viral load suppression, in addition to the variables men- mal liver enzymes off of antiretrovirals, and assessment for cir- tioned above, was associated with any of the markers in a sub- rhosis (no underlying liver injury etiology provided and liver set of patients with either HIV or HIV/HCV. All P values were tissue was normal). Active or recent alcohol use, evidence of adjusted using the Benjamini-Hochberg procedure for multiple nonalcoholic steatohepatitis (NASH) on biopsy with Brunt stage comparisons. 2 or greater fibrosis, and active HBV infection (+HBsAg) were RESULTS excluded. Control liver tissue is acquired from localized colorec- tal cancer tumor metastasis resection as part of a research tissue Overall, 66 liver specimens were identified: 5 controls, 7 HIV- biorepository protocol at Duke. Histologically normal tissue that infected patients, 27 HCV-infected patients, and 27 HIV/ is >1  cm from the tumor mass is collected for future use. HIV HCV-coinfected patients. Two HCV-infected subjects did not viral suppression was defined as RNA <200 copies/mL. have adequate tissue on staining and were therefore removed from the analysis. The controls were predominantly female Immunohistochemistry (80%), and the mean age at the time of liver biopsy was 52 years De-identified FFPE liver tissue sections were stained with anti- (±10.7). Race was only available for 1 of the controls, who was bodies against (1) key components of the Hh pathway, including white. The final analysis cohort (n  =  59) with demographic Sonic Hedgehog (SHH) ligand, glioblastoma 2 (GLI2) tran- data was 44% female and 54% black, with a mean age at the scription factor, and patched (PTCH1) receptor to assess liver time of the liver biopsy (SD) of 46 (8.7) years (Supplementary repair; (2) resident T-lymphocytes (CD3, CD8, CD56, CD28), Table  2). Thirty-four percent of all patients with HCV infec- CXCL16 (NKT cell cytokine); and (3) markers of immune acti- tion had severe liver fibrosis (METAVIR stage 3 or 4), and the vation and cellular aging (CD57 and p16INK4a). In diseased majority (92%) had genotype-1 infection. For all patients with liver tissue, CD56 may also be expressed by reactive bile ducts. HIV infection, the median CD4 (SD) was 651 (386) cells/mm , Please see Supplementary Table  1 for commercial suppliers and 85% were on antiretrovirals at the time of liver biopsy. Ten of the antibodies used. Staining for the myofibroblast marker patients (17%) had diabetes. alpha-smooth muscle actin (αSMA) was also performed. In e 3 inf Th ected cohorts (HIV, HCV, HIV/HCV) had addition, isotype/negative controls were completed for each greater expression of markers of tissue repair and fibrogene- antibody to ensure that positive staining is due to the antibody sis (GLI2 P  <  .0001, Shh P  <  .0001, PTCH1 P  =  .0214, αSMA binding of the desired antigen and not to some general unspe- P  <  .0001), T-cell populations (CD3 P  <  .0001), NK-like cells cific binding of the immunoglobulin to the tissue. (CD56 P  <  .0001), NKT cell recruitment cytokine (CXCL16 Morphometric analysis was completed with MetaView sowa ft re P  <  .0001), and markers of cellular senescence/aging (CD57 (Universal Imaging Corp, Downington, PA), and morphomet- INK4a P  =  .0003 and p16 P  <  .0001) than controls (Figures  1 ric quantification was done with 50 randomly chosen, 20x fields and 2; Supplementary Figure 1). We observed increased stain- per section. Immunoreactive cells were quantified using similar ing for the Hh pathway transcription factor GLI2 and ligand approaches for each of these stains; the numbers of cells with SHH in all 3 virally infected cohorts (Figure 1A and B). A rep- stained nuclei (GLI2) and stained membranes (immune markers, resentative immunohistochemistry section to show staining cytokines) were counted in 20 random, 20x fields per section. of GLI2 and SHH across all cohorts is provided in Figure  1C. Statistical Analysis Expression of tissue markers was greatest in HIV/HCV coin- The Student t test and analysis of variance were used to compare fection for PTCH1 (P = .016), CD3 (P = .028), CD57 (P = .017), ink4a histologic changes across the study groups, with significance set CXCL16 (P  =  .0002), and p16 (P  =  .096). Increased T-cell at a P value of <.05. To test for independent associations for each activation marker (CD57) staining was also observed for all 3 immunohistochemistry marker, multivariate linear regression virally infected cohorts, with the greatest staining in patients 2 • OFID • BRIEF REPORT Downloaded from https://academic.oup.com/ofid/article-abstract/5/7/ofy138/5035423 by Ed 'DeepDyve' Gillespie user on 16 October 2019 A B F 13.64 Prob > F <.0001 F 9.44 Prob > F <.0001 P = .236 P = .182 P = .011 P < .0001 40 P = .007 P = .006 Con HIV HCV HIV/HCV Con HIV HCV HIV/HCV C HCV Con HIV HIV/HCV Ductular Immune Figure 1. Hedgehog (Hh) activation markers in hepatic parenchyma of healthy and virally infected patients. Viral infection is associated with activation of the Hh pathway (GLI2 transcription factor and Sonic Hedgehog [SHH] ligand). Staining of liver tissue sections from controls (Con), patients with HIV infection (HIV), HCV infection (HCV), and HIV/HCV coinfection (HIV/HCV). Box and whisker plots of (A) GLI2 marker staining and (B) SHH marker staining across the 4 patient cohorts. C, Representative immunohisto- chemistry section with ductular and immune cell staining for GLI2 (brown) and ductular cell staining for SHH (green). with HIV/HCV coinfection (Figure  2A). Staining for the and increased cellular aging and αSMA markers. HIV/HCV αSMA fibrosis marker, shown in Figure  2B , is increased in coinfection had an additive impact, above and beyond that of HIV-monoinfected patients as compared with controls and is HIV or HCV alone, on levels of staining for SHH, CD3, CD8, ink4a greatest for patients with HCV and HIV/HCV infection. A rep- CD56, and p16 and trended toward significance (P  < .1) for resentative immunohistochemistry section to show staining for CD57 and CXCL16. In the subset of patients with either HIV or CD57, NKT cell marker (CD56), and αSMA across all cohorts HIV/HCV, HIV viral load suppression was not associated with is provided in Figure  2C and D. In addition, several markers any of the markers. were associated with elevated liver enzymes, including PTCH1 DISCUSSION (r = .411, P = .0017), CD56 (r = .421, P = .0011), CD57 (r = .297, P  =  .025) with AST, and CD56 (r  =  .286, P  =  .031) and CD28 HIV/HCV coinfection accelerates progressive liver fibrosis, (r = .264, P = .047) with ALT. yet the mechanisms remain poorly understood. The data, pre- Aer ft adjusting for clinical and demographic variables, the sented here for the first time, support a role for Hh activity in multivariate models identified METAVIR stage, HIV infection, HIV-related liver disease. Furthermore, the evidence of Hh acti- HCV infection, and the HIV/HCV coinfection interaction term vation even in HIV-infected patients without HCV coinfection as the primary independent predictors (P  <  .05) of increased suggests a potential role for ongoing liver fibrosis after HCV marker staining (Supplementary Table  3). As expected based eradication or a risk of fibrosis in the absence of HCV infection. on prior studies, METAVIR fibrosis stage was independently There is an increasing concern for progressive fibrogenesis after associated with Hh activation markers, increased T-cell mark- HCV eradication, with a recent study suggesting that a subset ers (activity and senescence), and increased αSMA staining. of HIV-infected patients will not normalize liver enzymes after HIV infection was independently associated with Hh activation HCV eradication [14]. It is critical to gain a more comprehen- markers, increased T-cell markers (activity, recruitment, and sive understanding of the active profibrogenic pathways in senescence), and increased cellular aging markers. HCV infec- HIV/HCV coinfection and to develop serologic biomarkers of tion was independently associated with Hh activation markers, fibrogenic risk with the long-term goal of targeting these path- increased T-cell markers (activity, recruitment, and senescence), ways for novel therapeutics. BRIEF REPORT • OFID • 3 GL12 marker staining (quantification=count) GL12 marker staining (quantification=count) Downloaded from https://academic.oup.com/ofid/article-abstract/5/7/ofy138/5035423 by Ed 'DeepDyve' Gillespie user on 16 October 2019 A B F 7.19 F 15.42 Prob > F 0.0003 Prob > F <.0001 P = .93 P = .017 P < .0001 P < .0001 P = .026 P = .002 Con HIV HCV HIV/HCV Con HIV HCV HIV/HCV Con HIV HCV HIV/HCV Immune Immune Stromal/HSC/MF Hepatocyte Ductular Ductular Figure  2. Immune activation and fibrosis markers in hepatic parenchyma of healthy and virally infected patients. Viral infection is associated with immune activation (CD57), NKT cell inflammation (CD56) and fibrogenesis (αSMA). Staining of liver tissue sections from controls (Con), patients with HIV infection (HIV), HCV infection (HCV), and HIV/HCV coinfection (HIV/HCV). Box and whisker plots of (A) CD57 T-cell terminal differentiation marker staining and (B) αSMA fibrosis marker staining across the 4 patient cohorts. C, Representative immunohistochemistry section with immune cell staining for CD57 (brown). D, Representative immunohistochemistry section with immune and ductular cell staining for CD56 (brown) and ductular and stromal (hepatic stellate cells, myofibroblasts) cell staining for αSMA (green). e e Th vidence for a role of Hh activation in human liver dis- (GLI2, SHH, and PTCH). Our data also indicate that fibrosis is ease includes nonalcoholic steatohepatitis (NASH), hepatitis B independently associated with T-cell populations in the liver, and C virus infections, schistosomiasis, and hepatocellular car- including NKT cells and CXCL16, a cytokine associated with cinoma [12, 15]. In animal models of NASH, Hh-responsive NKT cell recruitment and retention in liver tissue. Lastly, we HSCs undergo epithelial-to-mesenchymal transition into show that pathologic b fi rosis stage is independently associated myob fi roblasts, which acquire a prob fi rogenic phenotype [10]. with T-cell terminal differentiation, a marker of chronic immune A similar transition was recently reported in animal models of activation and senescence of the T cells in liver tissue. HIV-associated nephropathy, for the first time implicating Hh We report for the first time that liver tissue from patients with signaling in HIV-associated kidney fibrosis [9 ]. Hh ligands also HIV infection exhibits markers of liver tissue repair (Hh sig- upregulate expression of chemokines and receptors in immature naling), T-cell populations including NKT cells, and markers liver epithelial cells, thus facilitating hepatic recruitment and of cellular aging and senescence, independent of HCV coin- retention of immune cells known to play a key role in fibrogen- fection. In addition, we have confirmed prior work that HCV esis [12]. Here we confirm, in the setting of 2 chronic viral infection is independently associated with liver tissue repair, infections, that the pathologic stage of fibrosis on liver biopsy T-cell recruitment, and markers of fibrogenesis. The additive is independently associated with activation of the Hh pathway effect of HIV and HCV coinfection was evident for a majority of 4 • OFID • BRIEF REPORT GL12 marker staining (quantification=count) αSMA marker staining (quantification=count) Downloaded from https://academic.oup.com/ofid/article-abstract/5/7/ofy138/5035423 by Ed 'DeepDyve' Gillespie user on 16 October 2019 Acknowledgments the markers, suggesting that patients with HIV/HCV coinfec- Prior presentations. This work was presented in part at the European tion have a greater wound healing response to tissue injury and Association for the Study of Liver Disease Meeting; April 2015; Vienna, more severe immune dysregulation, which would be expected Austria. to translate to a greater risk of developing fibrosis. Financial support. This work was supported by the National Institute of Allergy and Infectious Diseases K23-AI096913 and Duke Center for Limitations of the current study include the use of preexisting AIDS Research (AI064518) to S.N.; Department of Veteran Affairs (Ralph clinical samples, which resulted in limited numbers of patients; H Johnson VAMC, Charleston) and MUSC GI Division Start-Up Funds thus these preliminary data will need to be confirmed in a larger (MUCU-2220300-35876-6125-00) to W.S. study. In addition, the cross-sectional and retrospective nature of Potential conflicts of interest. Dr. Diehl reports receiving consulting fees from Pfizer, Novartis, Boehringer Ingelheim, Allergan, Celgene, and the study and access only to fixed formalin tissue did not allow Lumena; participating in clinical trials with Allergan, Gilead Sciences, for more extensive investigations into mechanism; thus we can- Conatus, Galmed, NGM Biopharmaceuticals, Bristol-Myers Squibb, not comment on causation. Although we excluded any HIV- Madrigal, Galectin Therapeutics, Exalenz Biosciences, Shire, Intercept, Immuron, Boehringer Ingelheim, and Genfit; receiving grant support monoinfected patients with grossly abnormal liver tissue, there from and participating in a clinical trial with Immuron; receiving grant is likely selection bias in those who underwent liver biopsy in support for research collaborations from Metabolon, Prometheus, and the first place because liver biopsies in HIV monoinfection are Celgene; and holding a pending patent application for “Development of Novel Therapeutics to Treat Non-Alcoholic Steatohepatitis (NASH).” Dr. uncommon in clinical practice, and they were primarily done Naggie reports research support in the form of clinical trials or grant for elevated liver enzymes and/or abnormal imaging. To limit funding from AbbVie, Gilead, Janssen, Bristol Meyers Squibb, Tacere, the potential impact of nonalcoholic steatohepatitis, we excluded and Merck. HIV-infected patients with significant fibrosis, which limited our ability to assess the relationship of Hh signaling and immune acti- References vation with fibrosis in the HIV-infected cohort. Similarly, we failed 1. Smith CJ, Ryom L, Weber R, et  al; D:A:D Study Group. Trends in underlying causes of death in people with HIV from 1999 to 2011 (D:A:D): a multicohort to show that HIV/HCV coinfection has independently associated collaboration. Lancet 2014; 384:241–8. with the fibrogenesis marker αSMA. This was due to our study 2. Thein HH, Yi Q, Dore GJ, Krahn MD. Natural history of hepatitis C virus infec- tion in HIV-infected individuals and the impact of HIV in the era of highly active design, which matched HCV-infected and HIV/HCV-coinfected antiretroviral therapy: a meta-analysis. AIDS 2008; 22:1979–91. patients by severity of pathologic fibrosis stage. Further studies 3. Lin W, Weinberg EM, Chung RT. Pathogenesis of accelerated fibrosis in HIV/ HCV co-infection. J Infect Dis 2013; 207(Suppl 1):S13–8. will be conducted to address these potential limitations. 4. Salloum S, Holmes JA, Jindal R, et  al. Exposure to human immunodeficiency In conclusion, this is the first study to assess the role of the virus/hepatitis C virus in hepatic and stellate cell lines reveals cooperative profi- brotic transcriptional activation between viruses and cell types. Hepatology 2016; Hh signaling pathway in HIV-associated liver disease. Compared 64:1951–68. with controls, and independent of HCV coinfection, patients 5. Guy CD, Suzuki A, Zdanowicz M, et al; NASH CRN. Hedgehog pathway activa- with HIV infection had increased Hh pathway activity, increased tion parallels histologic severity of injury and fibrosis in human nonalcoholic fatty liver disease. Hepatology 2012; 55:1711–21. markers of T-cell-mediated inflammation, chronic activation, 6. Braune J, Weyer U, Matz-Soja M, et  al. Hedgehog signalling in myeloid cells and cellular aging. 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Exp Cell Res 2017; and increased cellular aging markers support a role for immune 352:193–201. dysfunction that may be promoted by differential constraint on 10. Choi SS, Omenetti A, Syn WK, Diehl AM. The role of Hedgehog signaling in fibrogenic liver repair. Int J Biochem Cell Biol 2011; 43:238–44. the Hh pathway noted across the different viral infection cohorts. 11. Beachy PA, Karhadkar SS, Berman DM. Tissue repair and stem cell renewal in Additional studies are needed to confirm our findings and fur - carcinogenesis. Nature 2004; 432:324–31. 12. Syn WK, Witek RP, Curbishley SM, et al. Role for hedgehog pathway in regulating ther elucidate the causal relationship of Hh pathway activation growth and function of invariant NKT cells. Eur J Immunol 2009; 39:1879–92. and T-cell recruitment, retention, and exhaustion. 13. Furler RL, Uittenbogaart CH. GLI2 regulates TGF-β1 in human CD4+ T cells: implications in cancer and HIV pathogenesis. PLoS One 2012; 7:e40874. 14. Hadigan CM, Howard L, Sheehan J, et  al. Persistent aminotransferase elevation Supplementary Data following HCV clearance in adults with and without HIV. Paper presented at: Supplementary materials are available at Open Forum Infectious Diseases 18th International Workshop on Co-morbidities and Adverse Drug Reactions in online. Consisting of data provided by the authors to benefit the reader, HIV; 12–13 September 2016; New York, NY. the posted materials are not copyedited and are the sole responsibility of 15. Pereira Tde A, Witek RP, Syn WK, et al. Viral factors induce Hedgehog pathway the authors, so questions or comments should be addressed to the corre- activation in humans with viral hepatitis, cirrhosis, and hepatocellular carcinoma. sponding author. Lab Invest 2010; 90:1690–703. BRIEF REPORT • OFID • 5 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Open Forum Infectious Diseases Oxford University Press

Markers of Tissue Repair and Cellular Aging Are Increased in the Liver Tissue of Patients With HIV Infection Regardless of Presence of HCV Coinfection

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Downloaded from https://academic.oup.com/ofid/article-abstract/5/7/ofy138/5035423 by Ed 'DeepDyve' Gillespie user on 16 October 2019 Open Forum Infectious Diseases BRIEF REPORT activation of hepatic stellate cells (HSCs) via the Toll-like recep- Markers of Tissue Repair and Cellular tor 4, cysteine-X-cysteine receptor 4, and chemokine motif Aging Are Increased in the Liver receptor 5 receptors [3, 4]. Tissue of Patients With HIV Infection Fibrosis is a wound-healing response to tissue (epithe- lial) injury. The regenerative responses of wound-healing dif- Regardless of Presence of HCV fer depending on the severity and chronicity of liver injury. Coinfection Although initially beneficial, the repair process can become 1,2 3 1,4 2 Susanna Naggie, Marzena Swiderska-Syn, Steve Choi, Sam Lusk, pathogenic if it continues aberrantly, leading to considerable 5 1 3,6 1 1 Audrey Lan, Guido Ferrari, Wing-Kin Syn, Cynthia D. Guy, and Anna Mae Diehl tissue remodeling and scar. The fetal morphogen hedgehog 1 2 Duke University School of Medicine, Durham, North Carolina; Duke Clinical Research 3 (Hh) signaling pathway has been implicated in multiple met- Institute, Durham, North Carolina; Medical University of South Carolina, Charleston, South 4 5 Carolina; Durham VA Medical Center, Durham, North Carolina; University of North Carolina abolic diseases, including nonalcoholic steatohepatitis, obesity, School of Medicine, Chapel Hill, North Carolina; Ralph H. Johnson VA Medical Center, and diabetes [5–7]. The Hh pathway has also been implicated Charleston, South Carolina in HCV mono-infection, which induces hepatic steatosis and insulin resistance [8], and now for the first time the Hh pathway Liver disease is a leading cause of HIV-related mortality. Hepatitis has been implicated in HIV-associated nephropathy [9]. C virus (HCV)–related fibrogenesis is accelerated in the setting of HIV coinfection, yet the mechanisms underlying this aggres- e Hh p Th athway, originally identified in Drosophilia , is a sive pathogenesis are unclear. We identified formalin-fixed paraf- highly conserved signaling pathway that orchestrates multiple fin-embedded liver tissue for HIV-infected patients, HCV-infected aspects of organogenesis and tissue remodeling [10]. Hh path- patients, HIV/HCV-coinfected patients, and controls at Duke way activation typically enhances the growth and viability of University Medical Center. De-identified sections were stained for Hh-responsive cells, whereas abrogating Hh signal transduction markers against the wound repair Hedgehog (Hh) pathway, res- usually triggers apoptosis, conferring a selective advantage for ident T-lymphocytes, and immune activation and cellular aging. cell types capable of responding to Hh [11]. Hh-ligands upreg- HIV infection was independently associated with Hh activation ulate expression of chemokines and receptors in immature and markers of immune dysregulation in the liver tissue. liver epithelial cells, thus facilitating hepatic recruitment and Keywords. b fi rogenesis; GLI; Hedgehog; hepatitis C virus; retention of particular types of immune cells [12]. Specifically, human immunodeficiency virus; patched; pathogenesis; Sonic activated NKT cells are recruited, and by avoiding apoptosis, Hedgehog; wound repair. they accumulate in liver tissue, in turn enhancing liver disease progression. Gli2, a Hh pathway transcription factor, is also reported to regulate transcription of TGFß-1, a key prob fi ro- Liver disease is a leading cause of HIV-related morbidity and genic cytokine implicated in HIV/HCV-related fibrogenesis mortality [1]. In the United States, chronic hepatitis C virus [13]. (HCV) infection is the leading cause of liver disease and related Using liver tissue specimens from HIV-infected patients, mortality in HIV infection [1]. HCV is a metabolically active HCV-infected patients, HIV/HCV-coinfected patients, and con- virus that, in the setting of HIV coinfection, results in acceler- trols, we assessed for markers of the Hh pathway, T-lymphocyte ated liver disease pathogenesis [2]. The mechanisms identified cellular subpopulations, cellular aging and senescence, and as potentially contributing to this more severe disease pathogen- b fi rogenesis. We proposed testing the hypothesis that Hh sig- esis include increased oxidative stress and downstream induc- naling is aberrantly activated in the liver tissue of HIV-infected tion of prob fi rotic pathways by cytokines including TGFß-1 and patients with HCV and that this increased Hh activity correlates with increased liver NK T-cell populations and increased mark- Received 13 March 2018; editorial decision 30 May 2018; accepted 8 June 2018; published ers of fibrosis. online June 11, 2018. Correspondence: S. Naggie, MD, MHS, Infectious Diseases Section, Duke Clinical Research Institute, 2400 Pratt Street, Durham, NC, 27705 (susanna.naggie@duke.edu). METHODS Open Forum Infectious Diseases © The Author(s) 2018. Published by Oxford University Press on behalf of Infectious Diseases Study Population Society of America. This is an Open Access article distributed under the terms of the Creative Formalin-fixed paraffin-embedded (FFPE) liver tissue sections Commons Attribution-NonCommercial-NoDerivs licence (http://creativecommons.org/licenses/ by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any were identified for HIV-infected patients, HCV-infected patients, medium, provided the original work is not altered or transformed in any way, and that the work HIV/HCV-coinfected patients, and de-identified controls from is properly cited. For commercial re-use, please contact journals.permissions@oup.com the Department of Pathology at Duke University Medical Center, DOI: 10.1093/ofid/ofy138 BRIEF REPORT • OFID • 1 Downloaded from https://academic.oup.com/ofid/article-abstract/5/7/ofy138/5035423 by Ed 'DeepDyve' Gillespie user on 16 October 2019 in accordance with National Institutes of Health (NIH) and models were built and adjusted for independent variables: sex, institutional guidelines for human subjects research. All HIV/ race, age, METAVIR fibrosis stage, METAVIR inflammation HCV-coinfected patients with available liver biopsy specimens score, steatosis, HIV infection, HCV infection, alanine ami- who did not have any exclusion criteria were included. HCV- notransferase (ALT), aspartate aminotransferase (AST), and monoinfected patients were then identified by matching to HIV/ diabetes. An interaction term was included to assess the effect HCV-coinfected patients by age (±5  years) at the time of liver of HIV/HCV coinfection when compared with monoinfection. biopsy, sex, and METAVIR stage (0–1 and 2–4) of liver fibrosis If the HIV/HCV coinfection interaction term is reported as on the biopsy. Due to the small number, all HIV-monoinfected significant, it suggests that HIV/HCV coinfection has a unique patients with available liver biopsy who did not have any exclu- impact on the staining of the marker of interest beyond that of sion criteria were included. Reasons provided on the requisition HIV or HCV monoinfection. Controls were not included in the form for liver biopsy for the HIV-monoinfected patients included regression models due to the lack of clinical data. Additional abnormal liver enzymes and steatosis noted on imaging (n = 4), multivariate linear regression models were used to determine ill-defined lesions on imaging (liver tissue was normal), abnor- if HIV viral load suppression, in addition to the variables men- mal liver enzymes off of antiretrovirals, and assessment for cir- tioned above, was associated with any of the markers in a sub- rhosis (no underlying liver injury etiology provided and liver set of patients with either HIV or HIV/HCV. All P values were tissue was normal). Active or recent alcohol use, evidence of adjusted using the Benjamini-Hochberg procedure for multiple nonalcoholic steatohepatitis (NASH) on biopsy with Brunt stage comparisons. 2 or greater fibrosis, and active HBV infection (+HBsAg) were RESULTS excluded. Control liver tissue is acquired from localized colorec- tal cancer tumor metastasis resection as part of a research tissue Overall, 66 liver specimens were identified: 5 controls, 7 HIV- biorepository protocol at Duke. Histologically normal tissue that infected patients, 27 HCV-infected patients, and 27 HIV/ is >1  cm from the tumor mass is collected for future use. HIV HCV-coinfected patients. Two HCV-infected subjects did not viral suppression was defined as RNA <200 copies/mL. have adequate tissue on staining and were therefore removed from the analysis. The controls were predominantly female Immunohistochemistry (80%), and the mean age at the time of liver biopsy was 52 years De-identified FFPE liver tissue sections were stained with anti- (±10.7). Race was only available for 1 of the controls, who was bodies against (1) key components of the Hh pathway, including white. The final analysis cohort (n  =  59) with demographic Sonic Hedgehog (SHH) ligand, glioblastoma 2 (GLI2) tran- data was 44% female and 54% black, with a mean age at the scription factor, and patched (PTCH1) receptor to assess liver time of the liver biopsy (SD) of 46 (8.7) years (Supplementary repair; (2) resident T-lymphocytes (CD3, CD8, CD56, CD28), Table  2). Thirty-four percent of all patients with HCV infec- CXCL16 (NKT cell cytokine); and (3) markers of immune acti- tion had severe liver fibrosis (METAVIR stage 3 or 4), and the vation and cellular aging (CD57 and p16INK4a). In diseased majority (92%) had genotype-1 infection. For all patients with liver tissue, CD56 may also be expressed by reactive bile ducts. HIV infection, the median CD4 (SD) was 651 (386) cells/mm , Please see Supplementary Table  1 for commercial suppliers and 85% were on antiretrovirals at the time of liver biopsy. Ten of the antibodies used. Staining for the myofibroblast marker patients (17%) had diabetes. alpha-smooth muscle actin (αSMA) was also performed. In e 3 inf Th ected cohorts (HIV, HCV, HIV/HCV) had addition, isotype/negative controls were completed for each greater expression of markers of tissue repair and fibrogene- antibody to ensure that positive staining is due to the antibody sis (GLI2 P  <  .0001, Shh P  <  .0001, PTCH1 P  =  .0214, αSMA binding of the desired antigen and not to some general unspe- P  <  .0001), T-cell populations (CD3 P  <  .0001), NK-like cells cific binding of the immunoglobulin to the tissue. (CD56 P  <  .0001), NKT cell recruitment cytokine (CXCL16 Morphometric analysis was completed with MetaView sowa ft re P  <  .0001), and markers of cellular senescence/aging (CD57 (Universal Imaging Corp, Downington, PA), and morphomet- INK4a P  =  .0003 and p16 P  <  .0001) than controls (Figures  1 ric quantification was done with 50 randomly chosen, 20x fields and 2; Supplementary Figure 1). We observed increased stain- per section. Immunoreactive cells were quantified using similar ing for the Hh pathway transcription factor GLI2 and ligand approaches for each of these stains; the numbers of cells with SHH in all 3 virally infected cohorts (Figure 1A and B). A rep- stained nuclei (GLI2) and stained membranes (immune markers, resentative immunohistochemistry section to show staining cytokines) were counted in 20 random, 20x fields per section. of GLI2 and SHH across all cohorts is provided in Figure  1C. Statistical Analysis Expression of tissue markers was greatest in HIV/HCV coin- The Student t test and analysis of variance were used to compare fection for PTCH1 (P = .016), CD3 (P = .028), CD57 (P = .017), ink4a histologic changes across the study groups, with significance set CXCL16 (P  =  .0002), and p16 (P  =  .096). Increased T-cell at a P value of <.05. To test for independent associations for each activation marker (CD57) staining was also observed for all 3 immunohistochemistry marker, multivariate linear regression virally infected cohorts, with the greatest staining in patients 2 • OFID • BRIEF REPORT Downloaded from https://academic.oup.com/ofid/article-abstract/5/7/ofy138/5035423 by Ed 'DeepDyve' Gillespie user on 16 October 2019 A B F 13.64 Prob > F <.0001 F 9.44 Prob > F <.0001 P = .236 P = .182 P = .011 P < .0001 40 P = .007 P = .006 Con HIV HCV HIV/HCV Con HIV HCV HIV/HCV C HCV Con HIV HIV/HCV Ductular Immune Figure 1. Hedgehog (Hh) activation markers in hepatic parenchyma of healthy and virally infected patients. Viral infection is associated with activation of the Hh pathway (GLI2 transcription factor and Sonic Hedgehog [SHH] ligand). Staining of liver tissue sections from controls (Con), patients with HIV infection (HIV), HCV infection (HCV), and HIV/HCV coinfection (HIV/HCV). Box and whisker plots of (A) GLI2 marker staining and (B) SHH marker staining across the 4 patient cohorts. C, Representative immunohisto- chemistry section with ductular and immune cell staining for GLI2 (brown) and ductular cell staining for SHH (green). with HIV/HCV coinfection (Figure  2A). Staining for the and increased cellular aging and αSMA markers. HIV/HCV αSMA fibrosis marker, shown in Figure  2B , is increased in coinfection had an additive impact, above and beyond that of HIV-monoinfected patients as compared with controls and is HIV or HCV alone, on levels of staining for SHH, CD3, CD8, ink4a greatest for patients with HCV and HIV/HCV infection. A rep- CD56, and p16 and trended toward significance (P  < .1) for resentative immunohistochemistry section to show staining for CD57 and CXCL16. In the subset of patients with either HIV or CD57, NKT cell marker (CD56), and αSMA across all cohorts HIV/HCV, HIV viral load suppression was not associated with is provided in Figure  2C and D. In addition, several markers any of the markers. were associated with elevated liver enzymes, including PTCH1 DISCUSSION (r = .411, P = .0017), CD56 (r = .421, P = .0011), CD57 (r = .297, P  =  .025) with AST, and CD56 (r  =  .286, P  =  .031) and CD28 HIV/HCV coinfection accelerates progressive liver fibrosis, (r = .264, P = .047) with ALT. yet the mechanisms remain poorly understood. The data, pre- Aer ft adjusting for clinical and demographic variables, the sented here for the first time, support a role for Hh activity in multivariate models identified METAVIR stage, HIV infection, HIV-related liver disease. Furthermore, the evidence of Hh acti- HCV infection, and the HIV/HCV coinfection interaction term vation even in HIV-infected patients without HCV coinfection as the primary independent predictors (P  <  .05) of increased suggests a potential role for ongoing liver fibrosis after HCV marker staining (Supplementary Table  3). As expected based eradication or a risk of fibrosis in the absence of HCV infection. on prior studies, METAVIR fibrosis stage was independently There is an increasing concern for progressive fibrogenesis after associated with Hh activation markers, increased T-cell mark- HCV eradication, with a recent study suggesting that a subset ers (activity and senescence), and increased αSMA staining. of HIV-infected patients will not normalize liver enzymes after HIV infection was independently associated with Hh activation HCV eradication [14]. It is critical to gain a more comprehen- markers, increased T-cell markers (activity, recruitment, and sive understanding of the active profibrogenic pathways in senescence), and increased cellular aging markers. HCV infec- HIV/HCV coinfection and to develop serologic biomarkers of tion was independently associated with Hh activation markers, fibrogenic risk with the long-term goal of targeting these path- increased T-cell markers (activity, recruitment, and senescence), ways for novel therapeutics. BRIEF REPORT • OFID • 3 GL12 marker staining (quantification=count) GL12 marker staining (quantification=count) Downloaded from https://academic.oup.com/ofid/article-abstract/5/7/ofy138/5035423 by Ed 'DeepDyve' Gillespie user on 16 October 2019 A B F 7.19 F 15.42 Prob > F 0.0003 Prob > F <.0001 P = .93 P = .017 P < .0001 P < .0001 P = .026 P = .002 Con HIV HCV HIV/HCV Con HIV HCV HIV/HCV Con HIV HCV HIV/HCV Immune Immune Stromal/HSC/MF Hepatocyte Ductular Ductular Figure  2. Immune activation and fibrosis markers in hepatic parenchyma of healthy and virally infected patients. Viral infection is associated with immune activation (CD57), NKT cell inflammation (CD56) and fibrogenesis (αSMA). Staining of liver tissue sections from controls (Con), patients with HIV infection (HIV), HCV infection (HCV), and HIV/HCV coinfection (HIV/HCV). Box and whisker plots of (A) CD57 T-cell terminal differentiation marker staining and (B) αSMA fibrosis marker staining across the 4 patient cohorts. C, Representative immunohistochemistry section with immune cell staining for CD57 (brown). D, Representative immunohistochemistry section with immune and ductular cell staining for CD56 (brown) and ductular and stromal (hepatic stellate cells, myofibroblasts) cell staining for αSMA (green). e e Th vidence for a role of Hh activation in human liver dis- (GLI2, SHH, and PTCH). Our data also indicate that fibrosis is ease includes nonalcoholic steatohepatitis (NASH), hepatitis B independently associated with T-cell populations in the liver, and C virus infections, schistosomiasis, and hepatocellular car- including NKT cells and CXCL16, a cytokine associated with cinoma [12, 15]. In animal models of NASH, Hh-responsive NKT cell recruitment and retention in liver tissue. Lastly, we HSCs undergo epithelial-to-mesenchymal transition into show that pathologic b fi rosis stage is independently associated myob fi roblasts, which acquire a prob fi rogenic phenotype [10]. with T-cell terminal differentiation, a marker of chronic immune A similar transition was recently reported in animal models of activation and senescence of the T cells in liver tissue. HIV-associated nephropathy, for the first time implicating Hh We report for the first time that liver tissue from patients with signaling in HIV-associated kidney fibrosis [9 ]. Hh ligands also HIV infection exhibits markers of liver tissue repair (Hh sig- upregulate expression of chemokines and receptors in immature naling), T-cell populations including NKT cells, and markers liver epithelial cells, thus facilitating hepatic recruitment and of cellular aging and senescence, independent of HCV coin- retention of immune cells known to play a key role in fibrogen- fection. In addition, we have confirmed prior work that HCV esis [12]. Here we confirm, in the setting of 2 chronic viral infection is independently associated with liver tissue repair, infections, that the pathologic stage of fibrosis on liver biopsy T-cell recruitment, and markers of fibrogenesis. The additive is independently associated with activation of the Hh pathway effect of HIV and HCV coinfection was evident for a majority of 4 • OFID • BRIEF REPORT GL12 marker staining (quantification=count) αSMA marker staining (quantification=count) Downloaded from https://academic.oup.com/ofid/article-abstract/5/7/ofy138/5035423 by Ed 'DeepDyve' Gillespie user on 16 October 2019 Acknowledgments the markers, suggesting that patients with HIV/HCV coinfec- Prior presentations. This work was presented in part at the European tion have a greater wound healing response to tissue injury and Association for the Study of Liver Disease Meeting; April 2015; Vienna, more severe immune dysregulation, which would be expected Austria. to translate to a greater risk of developing fibrosis. Financial support. This work was supported by the National Institute of Allergy and Infectious Diseases K23-AI096913 and Duke Center for Limitations of the current study include the use of preexisting AIDS Research (AI064518) to S.N.; Department of Veteran Affairs (Ralph clinical samples, which resulted in limited numbers of patients; H Johnson VAMC, Charleston) and MUSC GI Division Start-Up Funds thus these preliminary data will need to be confirmed in a larger (MUCU-2220300-35876-6125-00) to W.S. study. In addition, the cross-sectional and retrospective nature of Potential conflicts of interest. Dr. Diehl reports receiving consulting fees from Pfizer, Novartis, Boehringer Ingelheim, Allergan, Celgene, and the study and access only to fixed formalin tissue did not allow Lumena; participating in clinical trials with Allergan, Gilead Sciences, for more extensive investigations into mechanism; thus we can- Conatus, Galmed, NGM Biopharmaceuticals, Bristol-Myers Squibb, not comment on causation. Although we excluded any HIV- Madrigal, Galectin Therapeutics, Exalenz Biosciences, Shire, Intercept, Immuron, Boehringer Ingelheim, and Genfit; receiving grant support monoinfected patients with grossly abnormal liver tissue, there from and participating in a clinical trial with Immuron; receiving grant is likely selection bias in those who underwent liver biopsy in support for research collaborations from Metabolon, Prometheus, and the first place because liver biopsies in HIV monoinfection are Celgene; and holding a pending patent application for “Development of Novel Therapeutics to Treat Non-Alcoholic Steatohepatitis (NASH).” Dr. uncommon in clinical practice, and they were primarily done Naggie reports research support in the form of clinical trials or grant for elevated liver enzymes and/or abnormal imaging. To limit funding from AbbVie, Gilead, Janssen, Bristol Meyers Squibb, Tacere, the potential impact of nonalcoholic steatohepatitis, we excluded and Merck. 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Open Forum Infectious Diseases – Oxford University Press

Published: Jul 1, 2018

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