Faculty Bibliography

INTRODUCTION: Dendritic cells (DC) are professional antigen presenting cells (APC) that hold significant promise for the development of cancer vaccines. Endoplasmic reticular (ER)-stress can enhance the immunogenicity of APC. Since blockade of fatty acid synthesis increases ER-stress, we postulated that inhibition of fatty acid synthesis in DC would enhance their immune-stimulatory properties. METHODS: HumanmoDCwere generated fromPMBCby culture in GM-CSF/IL-4. Murine DC were generated from bone marrow using GM-CSF. TOFA-an acetyl-CoA carboxylase inhibitor-was used to block fatty acid synthesis. RESULTS: Blockade of fatty acid synthesis decreased DC development from precursors and mitigated DC proliferation in mice and humans. The mechanism was related to downregulation of Cyclin B-1-a pro-proliferative gene-and upregulation of Caspase 3 and PARP-apoptosis-related genes. Blockade of fatty acid synthesis enhanced DC activation and capacity for stimulation of immune effecter cells. TOFA-DC expressed elevated activated MAP-K, NF-kB, and PI3K/Akt intermediates and produced higher cytokines (Table). Peptide-pulsed TOFA-DC induced more vigorous proliferation of antigen-restricted CD4+ and CD8+ T cells, induced T cell IFN-g and TNF-a production, and enhanced CTL in vivo (Table). TOFA-DC induced higher NK expression of CD25 and production of IFN-g (Table). Accordingly, we found increased capacity for Notch signaling in TOFA-DC. TOFA-treated DC exhibited high ER-stress including increased expression of GRP-78, eIF2-a, p-eIF2-a and XBP-1, and blockade of ER-stress by a chaperone protein mitigated their enhanced immune-stimulation. (Table presented) CONCLUSIONS: Blockade of fatty acid synthesis markedly increases DC immunogenicity and capacity to induce CTL by augmenting ER stress. Our findings have significant implications for the design of immunotherapy vaccines

INTRODUCTION: Chronic pancreatitis (CP) and pancreatic cancer (PDAC) are characterized by an intense inflammatory infiltration. Regulation of this process is incompletely understood. We found that TLR7 expression is increased in CP and PDAC. We postulated that CP and PDAC depend on TLR7 signaling. METHODS: CP was induced in C57BL/6 mice using 7X daily caerulein injections (50mug/kg) for three weeks. KrasG12D mice which harbor a Kras mutation in pancreatic progenitor cells were used to model PDAC. TLR7 ligand (ssRNA40; 100mug/kg) was administered thrice-weekly. Blockade of TLR7 was accomplished with olignonucleotide inhibitors of TLR7 (IRS661). RESULTS: In CP and PDAC we observed substantially-increased TLR7-expression in both inflammatory and parenchymal cells, and significantly elevated levels of TLR7 ligands (Table). Ligation of TLR7 exacerbated CP in WT mice and accelerated tumor progression in KrasG12D mice (Table). TLR7 ligation altered epithelial cell expression of numerous oncogenic targets in PDAC including loss of PTEN, p16, and Cyclin D1 and up-regulation of p21, p27, p53, c-myc, SHPTP1, and TGF-beta. Further, TLR7 blockade protected against CP and pancreatic carcinogenesis, with marked reduction of inflammatory infiltration and complete arrest of tumor progression. TLR7 activation affected intra-pancreatic inflammation by interfacing with Notch pathway intermediates via STAT3 activation, and by inducing canonical NF-kB and MAP kinase signaling pathways- with corresponding blockade mitigating the pro-inflammatory and neoplastic effects of TLR7 activation. (Table presented) CONCLUSIONS: TLR7 ligation drives pancreatic inflammation and neoplasia via novel signaling mechanisms. Conversely, TLR7 blockade is powerfully protective. Targeting TLR7 holds promise for the treatment of CP and PDAC

Introduction: Chronic pancreatitis (CP) is characterized by fibro-inflammatory transformation of the pancreatic parenchyma. the precise cellular mediators in CP are incompletely understood. Dendritic cells (DC) have recently emerged as initiators of organ-specific inflammation. Pancreatic stellate cells (PSC) become potently pro-inflammatory in CP and are primarily responsible for deposition of extracellular matrix proteins and fibrillar collagen. We postulated that an intimate relationship between DC and PSC mediate the dramatic fibro-inflammatory changes in CP. Methods: DC were isolated from bone marrow aspirates and cultured for 8 days in complete RPMI supplemented with murine GMCSF. PSC were isolated aftermechanical and chemical digestion of pancreas, followed by density centrifugation and plastic adherence. DC-PSC co-culture was accomplished by culturing equal number of DC and PSC for 24 to 48 hours before washing off the non-adherent DC. Cell culture supernatant was assayed using cytometric bead or ELISA. for in vivo experiments, a three week caerulein model of chronic pancreatitis was employed in C57BL/6micewith selectmice receiving i. p. adoptive transfer of DC (1x106 thrice weekly). in vivo PSC activation was measured using monoclonal antibodies directed against Desmin and a-SMA. Results: in vitroDC-PSCco-culture resulted in activation ofPSCsurface phenotype and the production ofhigher levels ofPDGF,as well as numerous chemokines and cytokines byPSC(Table). Effectswere contingent on direct cellular interaction betweenDCand PSC as well as DCexpression of ICAM-1 and MyD88. in vivo i. p. adoptive transfer of DC in murine caerulein-induced chronic pancreatitis resulted in markedly increased fibro- inflammatory changes in the pancreas as well as 4-fold increase in percent desmin-positive acini, and approximately 80% a-SMA-positive acini (Figure). However, whereasDCinduced PSC to become pro-inflammatory, DC inhibited PSC proliferation, failed to stimulate PSC migration, and did not induce PSC to express higher levels of extra-cellular matrix proteins or Collagen I in co-culture experiments. Furthermore, our experimental data suggest bi-directional cross-talk asPSCprevented DCmaturation but acted as a potentDCchemoattractant. Conclusions: DCand PSC induce robust bidirectional crosstalk that affects CP. DCinduce PSC to become pro-inflammatory, but do cannot cause PSC to adopt fully mature myofibroblast-like properties. (Figure presented)

BACKGROUND & AIMS: The cellular mediators of acute pancreatitis are incompletely understood. Dendritic cells (DCs) can promote or suppress inflammation, depending on their subtype and context. We investigated the roles of DC in development of acute pancreatitis. METHODS: Acute pancreatitis was induced in CD11c.DTR mice using caerulein or L-arginine; DCs were depleted by administration of diphtheria toxin. Survival was analyzed using Kaplan-Meier method. RESULTS: Numbers of major histocompatibility complex II(+)CD11c(+) DCs increased 100-fold in pancreata of mice with acute pancreatitis to account for nearly 15% of intrapancreatic leukocytes. Intrapancreatic DCs acquired a distinct immune phenotype in mice with acute pancreatitis; they expressed higher levels of major histocompatibility complex II and CD86 and increased production of interleukin-6, membrane cofactor protein-1, and tumor necrosis factor-alpha. However, rather than inducing an organ-destructive inflammatory process, DCs were required for pancreatic viability; the exocrine pancreas died in mice that were depleted of DCs and challenged with caerulein or L-arginine. All mice with pancreatitis that were depleted of DCs died from acinar cell death within 4 days. Depletion of DCs from mice with pancreatitis resulted in neutrophil infiltration and increased levels of systemic markers of inflammation. However, the organ necrosis associated with depletion of DCs did not require infiltrating neutrophils, activation of nuclear factor-kappaB, or signaling by mitogen-activated protein kinase or tumor necrosis factor-alpha. CONCLUSIONS: DCs are required for pancreatic viability in mice with acute pancreatitis and might protect organs against cell stress

Acetaminophen (APAP) overdose is one of the most frequent causes of acute liver failure in the United States and is primarily mediated by toxic metabolites that accumulate in the liver upon depletion of glutathione stores. However, cells of the innate immune system, including natural killer (NK) cells, neutrophils, and Kupffer cells, have also been implicated in the centrilobular liver necrosis associated with APAP. We have recently shown that dendritic cells (DCs) regulate intrahepatic inflammation in chronic liver disease and, therefore, postulated that DC may also modulate the hepatotoxic effects of APAP. We found that DC immune-phenotype was markedly altered after APAP challenge. In particular, liver DC expressed higher MHC II, costimulatory molecules, and Toll-like receptors, and produced higher interleukin (IL)-6, macrophage chemoattractant protein-1 (MCP-1), and tumor necrosis factor alpha (TNF-alpha). Conversely, spleen DC were unaltered. However, APAP-induced centrilobular necrosis, and its associated mortality, was markedly exacerbated upon DC depletion. Conversely, endogenous DC expansion using FMS-like tyrosine kinase 3 ligand (Flt3L) protected mice from APAP injury. Our mechanistic studies showed that APAP liver DC had the particular capacity to prevent NK cell activation and induced neutrophil apoptosis. Nevertheless, the exacerbated hepatic injury in DC-depleted mice challenged with APAP was independent of NK cells and neutrophils or numerous immune modulatory cytokines and chemokines. Conclusion: Taken together, these data indicate that liver DC protect against APAP toxicity, whereas their depletion is associated with exacerbated hepatotoxicity. (HEPATOLOGY 2011;)

INTRODUCTION: Tolerance is the hallmark of hepatic immune function and dendritic cells (DC) are critical to this phenomenon. Lipid accumulation causes DC dysfunction in cancer.We postulated that liver DC are conspicuously lipid-rich and therefore mediate tolerance. METHODS: Immune cells from C57BL/6 livers were isolated using FACS. Cytokines were measured using a bead array. CTL lysis was measured using Cr51. NK and iNKT cells were tested in DC cocultures. EG7 was employed in tumor experiments. RESULTS: We discovered two distinct liver DC populations, high lipid containing (HL-DC) and low lipid containing (LL-DC). HL-DC exhibited an activated phenotype, produced robust levels of cytokines, activated CD4+ T, NK and iNKT cells, induced CTL responses and prevented tumor growth after adoptive transfer immunization. Conversely, LL-DC induced Tregs, anergy to cancer, and mediated tolerance to oral antigen. (Table Presented) CONCLUSIONS: Endogenous lipid content in hepatic DC subsets defines their immunogenic potential. Contrary to our hypothesis, HL-DC are potently immunogenic while LL-DC maintain tolerance. Manipulation of the HL-Dc: LL-DC ratio may be an attractive strategy for experimental therapeutics to modulate hepatic immunity and tolerance

The normal liver is characterized by immunologic tolerance. Primary mediators of hepatic immune tolerance are liver sinusoidal endothelial cells (LSECs). LSECs block adaptive immunogenic responses to Ag and induce the generation of T regulatory cells. Hepatic fibrosis is characterized by both intense intrahepatic inflammation and altered hepatic immunity. We postulated that, in liver fibrosis, a reversal of LSEC function from tolerogenic to proinflammatory and immunogenic may contribute to both the heightened inflammatory milieu and altered intrahepatic immunity. We found that, after fibrotic liver injury from hepatotoxins, LSECs become highly proinflammatory and secrete an array of cytokines and chemokines. In addition, LSECs gain enhanced capacity to capture Ag and induce T cell proliferation. Similarly, unlike LSECs in normal livers, in fibrosis, LSECs do not veto dendritic cell priming of T cells. Furthermore, whereas in normal livers, LSECs are active in the generation of T regulatory cells, in hepatic fibrosis LSECs induce an immunogenic T cell phenotype capable of enhancing endogenous CTLs and generating potent de novo CTL responses. Moreover, depletion of LSECs from fibrotic liver cultures mitigates the proinflammatory milieu characteristic of hepatic fibrosis. Our findings offer a critical understanding of the role of LSECs in modulating intrahepatic immunity and inflammation in fibro-inflammatory liver disease

The liver is the most common site of adenocarcinoma metastases, even in patients who initially present with early disease. We postulated that immune-suppressive cells in the liver of tumor-bearing hosts inhibit anti-tumor T cells, thereby accelerating the growth of liver metastases. Using models of early preinvasive pancreatic neoplasia and advanced colorectal cancer, aims of this study were to determine immune phenotype, stimulus for recruitment, inhibitory effects, and tumor-enabling function of immune-suppressive cells in the liver of tumor-bearing hosts. We found that in mice with intra-abdominal malignancies, two distinct CD11b(+)Gr1(+) populations with divergent phenotypic and functional properties accumulate in the liver, becoming the dominant hepatic leukocytes. Their expansion is contingent on tumor expression of KC. These cells are distinct from CD11b(+)Gr1(+) populations in other tissues of tumor-bearing hosts in terms of cellular phenotype and cytokine and chemokine profile. Liver CD11b(+)Gr1(+) cells are highly suppressive of T cell activation, proliferation, and cytotoxicity and induce the development of Tregs. Moreover, liver myeloid-derived suppressor cells accelerate the development of hepatic metastases by inactivation of cytotoxic T cells. These findings may explain the propensity of patients with intra-abdominal cancers to develop liver metastases and suggest a promising target for experimental therapeutics