Susan Specht

Immature and mature CD8α+ dendritic cells prolong the survival of vascularized heart allografts

CD8α+ and CD8α− dendritic cells (DCs) arise from committed bone marrow progenitors and can induce or regulate immune reactivity. Previously, the maturational status of CD8α− (myeloid) DCs has been shown to influence allogeneic T cell responses and allograft survival. Although CD8α+ DCs have been implicated in central tolerance and found to modulate peripheral T cell function, their influence on the outcome of organ transplantation has not been examined. Consistent with their equivalent high surface expression of MHC and costimulatory molecules, sorted mature C57BL/10J (B10; H2b) DCs of either subset primed naive, allogeneic C3H/HeJ (C3H; H2k) recipients for Th1 responses. Paradoxically and in contrast to their CD8α− counterparts, mature CD8α+ B10 DCs given systemically 7 days before transplant markedly prolonged B10 heart graft survival in C3H recipients. This effect was associated with specific impairment of ex vivo antidonor T cell proliferative responses, which was not reversed by exogenous IL-2. Further analyses of possible underlying mechanisms indicated that neither immune deviation nor induction of regulatory cells was a significant contributory factor. In contrast to the differential capacity of the mature DC subsets to affect graft outcome, immature CD8α+ and CD8α− DCs administered under the same experimental conditions significantly prolonged transplant survival. These observations demonstrate for the first time the innate capacity of CD8α+ DCs to regulate alloimmune reactivity and transplant survival, independent of their maturation status. Mobilization of such a donor DC subset with capacity to modulate antidonor immunity may have significant implications for the therapy of allograft rejection.

Gut‐derived commensal bacterial products inhibit liver dendritic cell maturation by stimulating hepatic interleukin‐6/signal transducer and activator of transcription 3 activity

Intraorgan dendritic cells (DCs) monitor the environment and help translate triggers of innate immunity into adaptive immune responses. Liver‐based DCs are continually exposed, via gut‐derived portal venous blood, to potential antigens and bacterial products that can trigger innate immunity. However, somehow the liver avoids a state of perpetual inflammation and protects central immune organs from overstimulation. In this study, we tested the hypothesis that hepatic interleukin‐6 (IL‐6)/signal transducer and activator of transcription 3 (STAT3) activity increases the activation/maturation threshold of hepatic DCs toward innate immune signals. The results show that the liver nuclear STAT3 activity is significantly higher than that of other organs and is IL‐6–dependent. Hepatic DCs in normal IL‐6 wild‐type (IL‐6+/+) mice are phenotypically and functionally less mature than DCs from IL‐6–deficient (IL‐6−/−) or STAT3‐inhibited IL‐6+/+ mice, as determined by surface marker expression, proinflammatory cytokine secretion, and allogeneic T‐cell stimulation. IL‐6+/+ liver DCs produce IL‐6 in response to exposure to lipopolysaccharide (LPS) and cytidine phosphate guanosine oligonucleotides (CpG) but are resistant to maturation compared with IL‐6−/− liver DCs. Conversely, exogenous IL‐6 inhibits LPS‐induced IL‐6−/− liver DC maturation. IL‐6/STAT3 signaling influences the liver DC expression of toll‐like receptor 9 and IL‐1 receptor associated kinase‐M. The depletion of gut commensal bacteria in IL‐6+/+ mice with oral antibiotics decreased portal blood endotoxin levels, lowered the expression of IL‐6 and phospho‐STAT3, and significantly increased liver DC maturation. Conclusion: Gut‐derived bacterial products, by stimulating hepatic IL‐6/STAT3 signaling, inhibit hepatic DC activation/maturation and thereby elevate the threshold needed for translating triggers of innate immunity into adaptive immune responses. Manipulating gut bacteria may therefore be an effective strategy for altering intrahepatic immune responses. (HEPATOLOGY 2007.)

Cooperation of p300 and PCAF in the control of microRNA 200c/141 transcription and epithelial characteristics.

Epithelial to mesenchymal transition (EMT) not only occurs during embryonic development and in response to injury, but is an important element in cancer progression. EMT and its reverse process, mesenchymal to epithelial transition (MET) is controlled by a network of transcriptional regulators and can be influenced by posttranscriptional and posttranslational modifications. EMT/MET involves many effectors that can activate and repress these transitions, often yielding a spectrum of cell phenotypes. Recent studies have shown that the miR-200 family and the transcriptional suppressor ZEB1 are important contributors to EMT. Our previous data showed that forced expression of SPRR2a was a powerful inducer of EMT and supports the findings by others that SPRR gene members are highly upregulated during epithelial remodeling in a variety of organs. Here, using SPRR2a cells, we characterize the role of acetyltransferases on the microRNA-200c/141 promoter and their effect on the epithelial/mesenchymal status of the cells. We show that the deacetylase inhibitor TSA as well as P300 and PCAF can cause a shift towards epithelial characteristics in HUCCT-1-SPRR2a cells. We demonstrate that both P300 and PCAF act as cofactors for ZEB1, forming a P300/PCAF/ZEB1 complex on the miR200c/141 promoter. This binding results in lysine acetylation of ZEB1 and a release of ZEB1 suppression on miR-200c/141 transcription. Furthermore, disruption of P300 and PCAF interactions dramatically down regulates miR-200c/141 promoter activity, indicating a PCAF/P300 cooperative function in regulating the transcriptional suppressor/activator role of ZEB1. These data demonstrate a novel mechanism of miRNA regulation in mediating cell phenotype.

Small proline-rich proteins 2 are noncoordinately upregulated by IL-6/STAT3 signaling after bile duct ligation.

Small proline-rich proteins 2 (SPRR2) are coordinately expressed with other epidermal differential complex (EDC) genes in the skin. They function as crosslinking proteins that form bridges between other proteins that comprise the cornified cell envelope, which is the major barrier against the environment. IL-6 is invariably produced at sites of biliary tract injury and IL-6-deficient (IL-6−/−) mice show impaired barrier function after bile duct ligation (BDL). Screening microarray analysis identified noncoordinate expression of SPRR2 as a candidate gene that is: (a) expressed in biliary epithelial cells (BEC); (b) IL-6 responsive; and (c) potentially related to biliary barrier function. Therefore, we studied in detail the regulation of BEC SPRR2A expression, in vitro; and tested the hypothesis that if BEC SPRR2 expression contributes to biliary barrier function, it should be increased after BDL in IL-6-wild type (IL-6+/+) mice and not in IL-6−/− mice. In vitro studies confirmed that IL-6/gp130-signaling, mediated primarily by signal transducer and activator of transcription 3 (STAT3), stimulated noncoordinate BEC SPRR2 expression. In vivo, noncoordinate upregulation of BEC SPRR2 expression after BDL was seen in the IL-6+/+ mice and was unrelated to squamous metaplasia. IL-6−/− mice showed deficient BEC SPRR2 expression after BDL associated with impaired barrier function, as evidenced by smaller diameters of obstructed ducts, decreased bile volume, and an inability to form ‘white bile’ compared to IL-6+/+ mice at 12 weeks after BDL. IL-6 replacement therapy reversed the barrier defect in IL-6−/− mice after BDL, coincident with recovery of SPRR2A expression. SPRR2 in diseased mouse and human liver localized subjacent to the apical plasma membrane of BEC lining bile ducts, but was more diffusely expressed throughout the cytoplasm of cholangioles. In conclusion, BEC IL-6/gp130/STAT3 signaling noncoordinately upregulates BEC SPRR2 that appears to contribute to modification of the biliary barrier under conditions of stress.

Wound Healing in the Biliary Tree of Liver Allografts

An increasing need for liver transplantation requires evaluation and triage of organs harvested from “extended criteria” donors. Although there is currently no widely accepted definition, most would agree that “extended criteria” includes organs donated by individuals that are old (>65 years), obese, infected with HBV or HCV, non-heart beating (NHBD), or had an unstable blood pressure before harvesting or the organ experienced a long cold ischemic time. These organs carry a statistical risk of dysfunction early after transplantation, but in the majority of recipients, hepatic parenchymal function recovers. Later, however, a small but significant percentage of extended criteria donors develop biliary strictures within several months after transplantation. The strictures occur primarily because of preservation injury that leads to “ischemic cholangitis” or deep wounding of the bile duct wall. Subsequent partial wound healing and wound contraction, but failed restitution of the biliary epithelial cell (BEC) lining, result in biliary tract strictures that cause progressive biliary fibrosis, increased morbidity, and decreased organ half-life. Better understanding of the pathophysiologic mechanisms that lead to biliary strictures in extended criteria donors provides an ideal proving ground for regenerative medicine; it also can provide insights into other diseases, such as extrahepatic biliary atresia and primary sclerosing cholangitis, that likely share certain pathogenic mechanisms. Possible points of therapeutic intervention include limiting cold and warm ischemic times, donor and/or donor organ treatment, ex vivo, to minimize the ischemic/preservation injury, maximize blood flow after transplantation, promote BEC wound healing, and limit myofibroblasts activation and proliferation in the bile duct wall. The pathobiology of biliary wound healing and therapeutic potential of interleukin-6 (IL-6) are highlighted.

Regulation and Function of Trefoil Factor Family 3 Expression in the Biliary Tree

Microarray analysis identified trefoil factor family 3 (TFF3) as a gene expressed in biliary epithelial cells (BECs), regulated by interleukin (IL)-6, and potentially involved in biliary pathophysiology. We therefore studied the regulation and function of BEC TFF3, in vitro and in vivo in IL-6(+/+) and IL-6(-/-) mice subjected to chronic bile duct ligation for 12 weeks. In vitro studies showed that IL-6 wild-type (IL-6(+/+)) BECs expressed higher TFF3 mRNA and protein levels than IL-6-deficient (IL-6(-/-)) BECs. BEC TFF3 expression is dependent primarily on signal transducer and activator of transcription (STAT3) signaling, but the reciprocal negative regulation known to exist between the intracellular IL-6/gp130 signaling pathways, STAT3 and mitogen-activated protein kinase (MAPK), importantly contributes to BEC TFF3 expression. Specifically blocking STAT3 activity with a dominant-negative molecule or treatment with a growth factor such as hepatocyte growth factor, which increases MAPK signaling, decreases BEC TFF3 expression. In contrast, specifically blocking MAPK activity with PD98059 significantly increased BEC TFF3 expression. Higher BEC TFF3 levels in IL-6(+/+) BECs were associated with significantly better migration than IL-6(-/-) BECs in a wound-healing assay and defective IL-6(-/-) BEC migration was reversed with exogenous TFF3. In vivo, hepatic TFF3 mRNA and protein expression was limited to BECs and dependent significantly on STAT3 signaling, but was influenced by other factors present after bile duct ligation. Comparable results were obtained in normal and diseased human tissue samples. In conclusion the regulation and function of BEC TFF3 expression is similar to the colon. BEC TFF3 expression depends primarily on gp130/STAT3 and contributes to BEC migration and wound healing. Therefore, use of recombinant IL-6 or TFF3 peptides should exert a therapeutic role in preventing biliary strictures in liver allografts.

Gut Bacteria Drive Kupffer Cell Expansion via MAMP-Mediated ICAM-1 Induction on Sinusoidal Endothelium and Influence Preservation-Reperfusion Injury after Orthotopic Liver Transplantation

Bacteria in the gut microbiome shed microbial-associated molecule patterns (MAMPs) into the portal venous circulation, where they augment various aspects of systemic immunity via low-level stimulation. Because the liver is immediately downstream of the intestines, we proposed that gut-derived MAMPs shape liver immunity and affect Kupffer cell (KC) phenotype. Germ-free (GF), antibiotic-treated (AVMN), and conventional (CL) mice were used to study KC development, function, and response to the significant stress of cold storage, reperfusion, and orthotopic transplantation. We found that a cocktail of physiologically active MAMPs translocate into the portal circulation, with flagellin (Toll-like receptor 5 ligand) being the most plentiful and capable of promoting hepatic monocyte influx in GF mice. In MAMP-deficient GF or AVMN livers, KCs are lower in numbers, have higher phagocytic activity, and have lower major histocompatibility complex II expression. MAMP-containing CL livers harbor significantly increased KC numbers via induction of intercellular adhesion molecule 1 on liver sinusoidal endothelium. These CL KCs have a primed yet expected phenotype, with increased major histocompatibility complex class II and lower phagocytic activity that increases susceptibility to liver preservation/reperfusion injury after orthotopic transplantation. The KC number, functional activity, and maturational status are directly related to the concentration of gut-derived MAMPs and can be significantly reduced by broad-spectrum antibiotics, thereby affecting susceptibility to injury.

Preexisting epithelial diversity in normal human livers: A tissue‐tethered cytometric analysis in portal/periportal epithelial cells

Routine light microscopy identifies two distinct epithelial cell populations in normal human livers: hepatocytes and biliary epithelial cells (BECs). Considerable epithelial diversity, however, arises during disease states when a variety of hepatocyte‐BEC hybrid cells appear. This has been attributed to activation and differentiation of putative hepatic progenitor cells (HPC) residing in the canals of Hering and/or metaplasia of preexisting mature epithelial cells. A novel analytic approach consisting of multiplex labeling, high‐resolution whole‐slide imaging (WSI), and automated image analysis was used to determine if more complex epithelial cell phenotypes preexist in normal adult human livers, which might provide an alternative explanation for disease‐induced epithelial diversity. “Virtually digested” WSI enabled quantitative cytometric analyses of individual cells displayed in a variety of formats (e.g., scatterplots) while still tethered to the WSI and tissue structure. We employed biomarkers specifically associated with mature epithelial forms (HNF4α for hepatocytes, CK19 and HNF1β for BEC) and explored for the presence of cells with hybrid biomarker phenotypes. The results showed abundant hybrid cells in portal bile duct BEC, canals of Hering, and immediate periportal hepatocytes. These bipotential cells likely serve as a reservoir for the epithelial diversity of ductular reactions, appearance of hepatocytes in bile ducts, and the rapid and fluid transition of BEC to hepatocytes, and vice versa. Conclusion: Novel imaging and computational tools enable increased information extraction from tissue samples and quantify the considerable preexistent hybrid epithelial diversity in normal human liver. This computationally enabled tissue analysis approach offers much broader potential beyond the results presented here. (HEPATOLOGY 2013)

Estrogen stimulates female biliary epithelial cell interleukin-6 expression in mice and humans†

Females are more susceptible than males to several biliary tract diseases. Interleukin‐6 (IL‐6) is critical to triggering autoimmune reactions and contributes substantially to biliary epithelial cell (BEC) barrier function and wound repair, and estrogen differentially regulates IL‐6 expression in various cell types. We hypothesized that estrogen might stimulate BEC IL‐6 production. Exposure to physiologic levels of estradiol, in vitro, increased female mouse BEC (mBEC) IL‐6 messenger RNA (mRNA) and protein expression, but either inhibited or had no effect on male mBECs. Female mBECs expressed higher concentrations of estrogen receptor‐alpha (ERα) mRNA and protein and were also more dependent on estradiol for survival, in vitro. In vivo, elevated estrogen during estrous cycling in mice, and estrogen treatment of mice harboring an ERα+ human cholangiocarcinoma resulted in increased BEC IL‐6 mRNA and tumor viability, respectively. Both responses could be blocked by an ERα antagonist. Human cholangiocarcinoma cell lines differentially expressing ERα were treated with specific ERα and ERβ agonists/antagonists to further test the relationship between estrogen stimulation, ERα expression, and IL‐6 production. Results show that ERα, and not the underlying BEC sex, was responsible for estrogen‐induced IL‐6 production. Estrogen‐induced proliferation of ERα‐expressing cholangiocarcinoma was blocked by anti–IL‐6 antibodies, indicating that at least some of the estrogen‐trophic effects are mediated via IL‐6. Finally, an association between ERα, IL‐6, and phosphorylated signal transducer and activator of transcription 3 (pSTAT3) signaling was shown in female‐predominant polycystic livers using immunohistochemical analyses, including multiplex quantum dot labeling. Conclusion: Estrogens stimulate IL‐6 production in non‐neoplastic female BECs and in neoplastic BECs expressing ERα. An association between these signaling pathways was demonstrated for female‐predominant polycystic livers and might also influence autoimmune hepatitis, primary biliary cirrhosis, and cholangiocarcinogenesis. (HEPATOLOGY 2010.)

Buccal mucosal cell immunohistochemistry: a simple method of determining the ABH phenotype of baboons, monkeys, and pigs

Abstract: Background: Baboons and monkeys fail to express ABH antigens on red blood cells (RBCs), and the A or H antigens are expressed only weakly on the surface of pig RBCs. Baboons and monkeys have been previously blood typed by detection of ABH antigens in the saliva after administration of pilocarpine. A reliable method to ABH type pigs is by immunohistochemical staining of renal distal tubules in kidney biopsies. We describe a simple and efficient method to blood type baboons, monkeys, and pigs.