Christopher S. Graffeo

Toll-like receptor 7 regulates pancreatic carcinogenesis in mice and humans

Pancreatic ductal adenocarcinoma is an aggressive cancer that interacts with stromal cells to produce a highly inflammatory tumor microenvironment that promotes tumor growth and invasiveness. The precise interplay between tumor and stroma remains poorly understood. TLRs mediate interactions between environmental stimuli and innate immunity and trigger proinflammatory signaling cascades. Our finding that TLR7 expression is upregulated in both epithelial and stromal compartments in human and murine pancreatic cancer led us to postulate that carcinogenesis is dependent on TLR7 signaling. In a mouse model of pancreatic cancer, TLR7 ligation vigorously accelerated tumor progression and induced loss of expression of PTEN, p16, and cyclin D1 and upregulation of p21, p27, p53, c-Myc, SHPTP1, TGF-β, PPARγ, and cyclin B1. Furthermore, TLR7 ligation induced STAT3 activation and interfaced with Notch as well as canonical NF-κB and MAP kinase pathways, but downregulated expression of Notch target genes. Moreover, blockade of TLR7 protected against carcinogenesis. Since pancreatic tumorigenesis requires stromal expansion, we proposed that TLR7 ligation modulates pancreatic cancer by driving stromal inflammation. Accordingly, we found that mice lacking TLR7 exclusively within their inflammatory cells were protected from neoplasia. These data suggest that targeting TLR7 holds promise for treatment of human pancreatic cancer.

Dendritic Cell Populations With Different Concentrations of Lipid Regulate Tolerance and Immunity in Mouse and Human Liver

BACKGROUND & AIMS Immune cells of the liver must be able to recognize and react to pathogens yet remain tolerant to food molecules and other nonpathogens. Dendritic cells (DCs) are believed to contribute to hepatic tolerance. Lipids have been implicated in dysfunction of DCs in cancer. Therefore, we investigated whether high lipid content in liver DCs affects induction of tolerance. METHODS Mouse and human hepatic nonparenchymal cells were isolated by mechanical and enzymatic digestion. DCs were purified by fluorescence-activated cell sorting or with immunomagnetic beads. DC lipid content was assessed by flow cytometry, immune fluorescence, and electron microscopy and by measuring intracellular component lipids. DC activation was determined from surface phenotype and cytokine profile. DC function was assessed in T-cell, natural killer (NK) cell, and NKT cell coculture assays as well as in vivo. RESULTS We observed 2 distinct populations of hepatic DCs in mice and humans based on their lipid content and expression of markers associated with adipogenesis and lipid metabolism. This lipid-based dichotomy in DCs was unique to the liver and specific to DCs compared with other hepatic immune cells. However, rather than mediate tolerance, the liver DC population with high concentrations of lipid was immunogenic in multiple models; they activated T cells, NK cells, and NKT cells. Conversely, liver DCs with low levels of lipid induced regulatory T cells, anergy to cancer, and oral tolerance. The immunogenicity of lipid-rich liver DCs required their secretion of tumor necrosis factor α and was directly related to their high lipid content; blocking DC synthesis of fatty acids or inhibiting adipogenesis (by reducing endoplasmic reticular stress) reduced DC immunogenicity. CONCLUSIONS Human and mouse hepatic DCs are composed of distinct populations that contain different concentrations of lipid, which regulates immunogenic versus tolerogenic responses in the liver.

Interleukin 17–Producing γδT Cells Promote Hepatic Regeneration in Mice

BACKGROUND & AIMS Subsets of leukocytes synergize with regenerative growth factors to promote hepatic regeneration. γδT cells are early responders to inflammation-induced injury in a number of contexts. We investigated the role of γδT cells in hepatic regeneration using mice with disruptions in Tcrd (encodes the T-cell receptor δ chain) and Clec7a (encodes C-type lectin domain family 7 member a, also known as DECTIN1). METHODS We performed partial hepatectomies on wild-type C57BL/6, CD45.1, Tcrd(-/-), or Clec7a(-/-) mice. Cells were isolated from livers of patients and mice via mechanical and enzymatic digestion. γδT cells were purified by fluorescence-activated cell sorting. RESULTS In mice, partial hepatectomy up-regulated expression of CCL20 and ligands of Dectin-1, which was associated with recruitment and activation of γδT cells and their increased production of interleukin (IL)-17 family cytokines. Recruited γδT cells induced production of IL-6 by antigen-presenting cells and suppressed expression of interferon gamma by natural killer T cells, promoting hepatocyte proliferation. Absence of IL-17-producing γδT cells or deletion of Dectin-1 prevented development of regenerative phenotypes in subsets of innate immune cells. This slowed liver regeneration and was associated with reduced expression of regenerative growth factors and cell cycle regulators. Conversely, exogenous administration of IL-17 family cytokines or Dectin-1 ligands promoted regeneration. More broadly, we found that γδT cells are required for inflammatory responses mediated by IL-17 and Dectin-1. CONCLUSIONS γδT cells regulate hepatic regeneration by producing IL-22 and IL-17, which have direct mitogenic effects on hepatocytes and promote a regenerative phenotype in hepatic leukocytes, respectively. Dectin-1 ligation is required for γδT cells to promote hepatic regeneration.

Divergent effects of RIP1 or RIP3 blockade in murine models of acute liver injury

Necroptosis is a recently described Caspase 8-independent method of cell death that denotes organized cellular necrosis. The roles of RIP1 and RIP3 in mediating hepatocyte death from acute liver injury are incompletely defined. Effects of necroptosis blockade were studied by separately targeting RIP1 and RIP3 in diverse murine models of acute liver injury. Blockade of necroptosis had disparate effects on disease outcome depending on the precise etiology of liver injury and component of the necrosome targeted. In ConA-induced autoimmune hepatitis, RIP3 deletion was protective, whereas RIP1 inhibition exacerbated disease, accelerated animal death, and was associated with increased hepatocyte apoptosis. Conversely, in acetaminophen-mediated liver injury, blockade of either RIP1 or RIP3 was protective and was associated with lower NLRP3 inflammasome activation. Our work highlights the fact that diverse modes of acute liver injury have differing requirements for RIP1 and RIP3; moreover, within a single injury model, RIP1 and RIP3 blockade can have diametrically opposite effects on tissue damage, suggesting that interference with distinct components of the necrosome must be considered separately.

Role of Fatty-Acid Synthesis in Dendritic Cell Generation and Function

Dendritic cells (DC) are professional APCs that regulate innate and adaptive immunity. The role of fatty-acid synthesis in DC development and function is uncertain. We found that blockade of fatty-acid synthesis markedly decreases dendropoiesis in the liver and in primary and secondary lymphoid organs in mice. Human DC development from PBMC precursors was also diminished by blockade of fatty-acid synthesis. This was associated with higher rates of apoptosis in precursor cells and increased expression of cleaved caspase-3 and BCL-xL and downregulation of cyclin B1. Further, blockade of fatty-acid synthesis decreased DC expression of MHC class II, ICAM-1, B7-1, and B7-2 but increased their production of selected proinflammatory cytokines including IL-12 and MCP-1. Accordingly, inhibition of fatty-acid synthesis enhanced DC capacity to activate allogeneic as well as Ag-restricted CD4+ and CD8+ T cells and induce CTL responses. Further, blockade of fatty-acid synthesis increased DC expression of Notch ligands and enhanced their ability to activate NK cell immune phenotype and IFN-γ production. Because endoplasmic reticulum (ER) stress can augment the immunogenic function of APC, we postulated that this may account for the higher DC immunogenicity. We found that inhibition of fatty-acid synthesis resulted in elevated expression of numerous markers of ER stress in humans and mice and was associated with increased MAPK and Akt signaling. Further, lowering ER stress by 4-phenylbutyrate mitigated the enhanced immune stimulation associated with fatty-acid synthesis blockade. Our findings elucidate the role of fatty-acid synthesis in DC development and function and have implications to the design of DC vaccines for immunotherapy.

A panoramic view of the skull base: systematic review of open and endoscopic endonasal approaches to four tumors.

Endoscopic endonasal surgery has been established as the safest approach to pituitary tumors, yet its role in other common skull base lesions has not been established. To answer this question, we carried out a systematic review of reported series of open and endoscopic endonasal approaches to four major skull base tumors: olfactory groove meningiomas (OGM), tuberculum sellae meningiomas (TSM), craniopharyngiomas (CRA), and clival chordomas (CHO). Data from 162 studies containing 5,701 patients were combined and compared for differences in perioperative mortality, gross total resection (GTR), cerebrospinal fluid (CSF) leak, neurological morbidity, post-operative visual function, post-operative anosmia, post-operative diabetes insipidus (DI), and post-operative obesity/hyperphagia. Weighted average rates for each outcome were calculated using relative study size. Our findings indicate similar rates of GTR and perioperative mortality between open and endoscopic approaches for all tumor types. CSF leak was increased after endoscopic surgery. Visual function symptoms were more likely to improve after endoscopic surgery for TSM, CRA, and CHO. Post-operative DI and obesity/hyperphagia were significantly increased after open resection in CRA. Recurrence rates per 1,000 patient-years of follow-up were higher in endoscopy for OGM, TSM, and CHO. Trends for open and endoscopic surgery suggested modest improvement in all outcomes over time. Our observations suggest that endonasal endoscopy is a safe alternative to craniotomy and may be preferred for certain tumor types. However, endoscopic surgery is associated with higher rates of CSF leak, and possibly increased recurrence rates. Prospective study with long-term follow-up is required to verify these preliminary observations.

Noninvasive diagnosis and management of spontaneous intracranial hypotension in patients with marfan syndrome: Case Report and Review of the Literature.

Background: Spontaneous intracranial hypotension is an uncommon clinical entity. Heritable connective tissue disorders (HCTD), such as Marfan syndrome, are frequently implicated as an underlying cause, due to dural structural weaknesses that predispose patients to spontaneous cerebrospinal fluid (CSF) leak. Due to the high prevalence of multi-system disease in HCTD, diagnosis and treatment are often complicated. Case Description: We present a 58-year-old female with Marfan syndrome on anticoagulation for a mechanical aortic valve replacement who came to medical attention with severe, acute-onset headache following a straining episode. Noninvasive magnetic resonance (MR) myelography confirmed thoracic CSF extravasations and multiple lumbar diverticula. The patient was treated conservatively and her symptoms resolved. Conclusion: We discuss the common presentation, diagnostic tools, and treatment options for spontaneous CSF leaks in patients with Marfan syndrome or related HCTD with an emphasis on noninvasive modalities and a review of the major radiographic criteria used to diagnose dural abnormalities, such as dural ectasia.

Abstract A102: Dendritic cells contribute to pancreatic fibroinflammatory disease and the transition to neoplasia.

Introduction: Chronic pancreatitis is the most prevalent risk factor for the development of pancreatic cancer. Dendritic cells (DC) are known to augment inflammation in a range of clinically significant contexts, yet their role in chronic pancreatitis and pancreatic cancer development has not been intensely studied. We postulated a primary role for DC in pancreatic inflammation and the transition to carcinogenesis. Methods: Chronic pancreatitis was induced using caerulein (50µg/kg, 3 weeks) in C57BL/6 mice. The p48Cre;Kras G12D model was used to study pancreatic carcinogenesis. Pancreatic DC populations were expanded by i.p. adoptive transfer of bone marrow-derived DC (1x106 cells, 3X/week) or using Flt3L (10µg/day x 10 days). MyD88 was inhibited in DC using CD11c-Cre MyD88 Floxed +/+ bone marrow chimeric p48Cre;Kras G12D mice. Results: DC accounted for 1-3% of CD45 + leukocytes in normal pancreata, but increased to 10-15% during chronic pancreatitis. Moreover, the absolute number of pancreatic DC increased 100-fold. The number of DC was similarly increased in the pancreata of p48Cre;Kras G12D mice compared with aged matched WT or p48Cre controls. Furthermore, the surface phenotype of DC infiltrating p48Cre;Kras G12D pancreata differed from controls in that they were highly mature, expressing elevated CD40 and CD86. DC adoptive transfer to mice experiencing chronic pancreatitis resulted in severely exacerbated fibro-inflammatory disease, including greater than 60% reduction in acinar cell volume and a marked reduction in islet cell mass. Further, mice treated with Flt3L and simultaneous challenge with caerulein showed exacerbated acinar destruction, fibrosis, and inflammation. In addition to the fibroinflammatory changes, DC-overexpansion in pancreatitis led to widespread development of early PanIN lesions. Overall, approximately 40% of ducts were classified as PanINs in mice adoptively transferred with DC. Moreover, DC markedly accelerated malignant transformation when transferred for four weeks to p48Cre;Kras G12D mice as evidenced by a two-fold increase in tumor weight. Protein analysis revealed that DC transfer resulted in altered pancreatic expression of numerous cell cycle regulatory and tumor suppressor genes including upregulated expression of p21, p27, p-p27, p53, and Rb. DC effects in pancreatic fibroinflammation and carcinogenesis were mediated by induction of Th2-deviated CD4 + T cells. The number of intra-pancreatic CD4 + T cells and CD4:CD8 T cell ratio was markedly increased in mice adoptively transferred with DC. Additionally, intra-pancreatic CD4 + T cells in DC-treated mice exhibited a strong Th2 differentiation. Accentuation of the DCTh2 axis by inhibiting MyD88 signaling in DC further accelerated carcinogenesis in p48Cre;Kras G12D mice. CD4 + T cell depletion, protected p48Cre;Kras G12D mice adoptively transferred with DC from developing accelerated carcinogenesis. Further, adoptive transfer of Th2-deviated CD4 + T cells from pancreatitic mice that had previously been transferred with DC markedly accelerated carcinogenesis. Conclusion: DC expand in chronic pancreatitis and pancreatic cancer. DC over-expansion in chronic pancreatitis exacerbates endocrine and exocrine destruction, induces marked inflammation, and early PanIN lesions. DC expansion in p48Cre;Kras G12D results in marked acceleration of tumor growth. DC effects on pancreatic fibroinflammation and neoplasia are mediated by induction of pancreatic-antigen-restricted Th2 cells. Citation Format: Saman Zarbakhsh, Harry Mushlin, Raghavendra Rao, Sanna Badar, Mohsin Jamal, Dafna Bar-Sagi, George Miller, Adeel Rehman, Christopher Graffeo, Atsuo Ochi, Rocky Barilla, Constantinos Zambirinis, Nina Fallon, Cristina Hajdu, Yuliya Pylayeva-Gupta. Dendritic cells contribute to pancreatic fibroinflammatory disease and the transition to neoplasia. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr A102.