Daniel Tippens

The necrosome promotes pancreatic oncogenesis via CXCL1 and Mincle-induced immune suppression

Neoplastic pancreatic epithelial cells are believed to die through caspase 8-dependent apoptotic cell death, and chemotherapy is thought to promote tumour apoptosis. Conversely, cancer cells often disrupt apoptosis to survive. Another type of programmed cell death is necroptosis (programmed necrosis), but its role in pancreatic ductal adenocarcinoma (PDA) is unclear. There are many potential inducers of necroptosis in PDA, including ligation of tumour necrosis factor receptor 1 (TNFR1), CD95, TNF-related apoptosis-inducing ligand (TRAIL) receptors, Toll-like receptors, reactive oxygen species, and chemotherapeutic drugs. Here we report that the principal components of the necrosome, receptor-interacting protein (RIP)1 and RIP3, are highly expressed in PDA and are further upregulated by the chemotherapy drug gemcitabine. Blockade of the necrosome in vitro promoted cancer cell proliferation and induced an aggressive oncogenic phenotype. By contrast, in vivo deletion of RIP3 or inhibition of RIP1 protected against oncogenic progression in mice and was associated with the development of a highly immunogenic myeloid and T cell infiltrate. The immune-suppressive tumour microenvironment associated with intact RIP1/RIP3 signalling depended in part on necroptosis-induced expression of the chemokine attractant CXCL1, and CXCL1 blockade protected against PDA. Moreover, cytoplasmic SAP130 (a subunit of the histone deacetylase complex) was expressed in PDA in a RIP1/RIP3-dependent manner, and Mincle—its cognate receptor—was upregulated in tumour-infiltrating myeloid cells. Ligation of Mincle by SAP130 promoted oncogenesis, whereas deletion of Mincle protected against oncogenesis and phenocopied the immunogenic reprogramming of the tumour microenvironment that was induced by RIP3 deletion. Cellular depletion suggested that whereas inhibitory macrophages promote tumorigenesis in PDA, they lose their immune-suppressive effects when RIP3 or Mincle is deleted. Accordingly, T cells, which are not protective against PDA progression in mice with intact RIP3 or Mincle signalling, are reprogrammed into indispensable mediators of anti-tumour immunity in the absence of RIP3 or Mincle. Our work describes parallel networks of necroptosis-induced CXCL1 and Mincle signalling that promote macrophage-induced adaptive immune suppression and thereby enable PDA progression.

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.

Dectin-1 Regulates Hepatic Fibrosis and Hepatocarcinogenesis by Suppressing TLR4 Signaling Pathways

Dectin-1 is a C-type lectin receptor critical in anti-fungal immunity, but Dectin-1 has not been linked to regulation of sterile inflammation or oncogenesis. We found that Dectin-1 expression is upregulated in hepatic fibrosis and liver cancer. However, Dectin-1 deletion exacerbates liver fibro-inflammatory disease and accelerates hepatocarcinogenesis. Mechanistically, we found that Dectin-1 protects against chronic liver disease by suppressing TLR4 signaling in hepatic inflammatory and stellate cells. Accordingly, Dectin-1(-/-) mice exhibited augmented cytokine production and reduced survival in lipopolysaccharide (LPS)-mediated sepsis, whereas Dectin-1 activation was protective. We showed that Dectin-1 inhibits TLR4 signaling by mitigating TLR4 and CD14 expression, which are regulated by Dectin-1-dependent macrophage colony stimulating factor (M-CSF) expression. Our study suggests that Dectin-1 is an attractive target for experimental therapeutics in hepatic fibrosis and neoplastic transformation. More broadly, our work deciphers critical cross-talk between pattern recognition receptors and implicates a role for Dectin-1 in suppression of sterile inflammation, inflammation-induced oncogenesis, and LPS-mediated sepsis.

Radiation Therapy Induces Macrophages to Suppress T-Cell Responses Against Pancreatic Tumors in Mice.

BACKGROUND & AIMS The role of radiation therapy in the treatment of patients with pancreatic ductal adenocarcinoma (PDA) is controversial. Randomized controlled trials investigating the efficacy of radiation therapy in patients with locally advanced unresectable PDA have reported mixed results, with effects ranging from modest benefit to worse outcomes compared with control therapies. We investigated whether radiation causes inflammatory cells to acquire an immune-suppressive phenotype that limits the therapeutic effects of radiation on invasive PDAs and accelerates progression of preinvasive foci. METHODS We investigated the effects of radiation therapy in p48(Cre);LSL-Kras(G12D) (KC) and p48(Cre);LSLKras(G12D);LSL-Trp53(R172H) (KPC) mice, as well as in C57BL/6 mice with orthotopic tumors grown from FC1242 cells derived from KPC mice. Some mice were given neutralizing antibodies against macrophage colony-stimulating factor 1 (CSF1 or MCSF) or F4/80. Pancreata were exposed to doses of radiation ranging from 2 to 12 Gy and analyzed by flow cytometry. RESULTS Pancreata of KC mice exposed to radiation had a higher frequency of advanced pancreatic intraepithelial lesions and more foci of invasive cancer than pancreata of unexposed mice (controls); radiation reduced survival time by more than 6 months. A greater proportion of macrophages from radiation treated invasive and preinvasive pancreatic tumors had an immune-suppressive, M2-like phenotype compared with control mice. Pancreata from mice exposed to radiation had fewer CD8(+) T cells than controls, and greater numbers of CD4(+) T cells of T-helper 2 and T-regulatory cell phenotypes. Adoptive transfer of T cells from irradiated PDA to tumors of control mice accelerated tumor growth. Radiation induced production of MCSF by PDA cells. A neutralizing antibody against MCSF prevented radiation from altering the phenotype of macrophages in tumors, increasing the anti-tumor T-cell response and slowing tumor growth. CONCLUSIONS Radiation treatment causes macrophages murine PDA to acquire an immune-suppressive phenotype and disabled T-cell-mediated anti-tumor responses. MCSF blockade negates this effect, allowing radiation to have increased efficacy in slowing tumor growth.

TGF-β Blockade Reduces Mortality and Metabolic Changes in a Validated Murine Model of Pancreatic Cancer Cachexia

Cancer cachexia is a debilitating condition characterized by a combination of anorexia, muscle wasting, weight loss, and malnutrition. This condition affects an overwhelming majority of patients with pancreatic cancer and is a primary cause of cancer-related death. However, few, if any, effective therapies exist for both treatment and prevention of this syndrome. In order to develop novel therapeutic strategies for pancreatic cancer cachexia, appropriate animal models are necessary. In this study, we developed and validated a syngeneic, metastatic, murine model of pancreatic cancer cachexia. Using our model, we investigated the ability of transforming growth factor beta (TGF-β) blockade to mitigate the metabolic changes associated with cachexia. We found that TGF-β inhibition using the anti-TGF-β antibody 1D11.16.8 significantly improved overall mortality, weight loss, fat mass, lean body mass, bone mineral density, and skeletal muscle proteolysis in mice harboring advanced pancreatic cancer. Other immunotherapeutic strategies we employed were not effective. Collectively, we validated a simplified but useful model of pancreatic cancer cachexia to investigate immunologic treatment strategies. In addition, we showed that TGF-β inhibition can decrease the metabolic changes associated with cancer cachexia and improve overall survival.

Abstract 3184: Tumor-entrained dendritic cells promote ICOS/ICOSL-dependent Th17-like responses in pancreatic adenocarcinoma

BACKGROUND: Ranking 4th among cancer-related deaths worldwide, pancreatic adenocarcinoma (PDAC) boasts a dismal prognosis. The robust immune infiltrate that comprises its tumor microenvironment (TME) can influence disease progression. Tumor rejection by T lymphocytes relies on proper guidance by dendritic cells (DCs). This antigen-education is manifested by the DCs’ expression of co-stimulatory factors, such as ICOS-ligand (ICOSL). In this study, we plan to elucidate the mechanisms used by PDAC-entrained DC to modify T cell effector function and differentiation. METHODS: To establish our in vivo model, either PBS or 2.5e+5 “FC1242” murine PDAC cells derived from the tumor of a KPC mouse (LSL-KrasG12D; Trp53R172H; pdxCre/+) were injected into the distal pancreata of C57BL/6J (WT) mice. After 20 days, mice were euthanized and the spleen and tumor were assayed. DC phenotype was determined by flow cytometry (FC). DC function was assessed by mixed-leukocyte reactions (MLR) and peptide-specific proliferation assays, +/- neutralizing mAbs against ICOSL or ICOS. Supernatant cytokines were assessed at 84h using cytometric bead assays (CBA). Intracellular cytokines were analyzed by FC after 5h re-stimulation with PMA/IMN. To assess in vivo proliferation, 2.5e+6 of both OT1 and OT2 T cells labeled with CFSE or CPD, respectively, were co-injected into WT mice and challenged with 5e+5 OVA-loaded DC after 24h. Proliferation was assessed 4 days post-DC inoculation. RESULTS: Splenic FC1242-DC had lower expression of MHC2 and CD83, while tumor-infiltrating DC (TME-DC) displayed higher levels of each, relative to controls. TME-DC also exhibited higher expression of ICOSL, CD80, and CD86. Despite no difference in ICOSL expression between splenic groups, MHC2 and CD83 were substantially lower on ICOSL+ DC of FC1242 spleen, while highest on that of the TME, relative to controls. We found a higher fraction of CD11b+ myeloid DC (mDC) and a lower fraction of cross-presenting CD8α+ DC (xDC) in both tumor-bearing groups relative to controls. FC1242-mDC expressed higher CD80 and PDL1 than sham-mDC, while FC1242-xDC displayed lower CD80 and no difference in PDL1. Also, tumor-entrained DC had a diminished propensity for CD8+ T cell expansion in vitro and in vivo, resulting in lower Granzyme B, IFNγ, and TNFα. Interestingly, DC tumor-entrainment improved CD4+ T cell expansion and augmented IL-17A, IL-6, G-CSF secretion, while diminishing IFNγ, TNFα, IL-13, and IL-2. Blockade of the ICOS-pathway hindered IL17 production, and amplified production of IFNγ and TNFα by T cells. CONCLUSION: This study suggests that DC possess a vital role as liaisons between the TME and cell-mediated immunity. Furthermore, Th17 cells in the TME can accelerate PDAC progression. This makes the ICOS-pathway especially attractive, as it can modify anti-tumor immunity while circumventing the barriers of drug-delivery to the TME. Citation Format: Rocky M. Barilla, Raul Caso, Antonina Avanzi, Anjlee Panjwani, Xiaopei L. Zeng, Steve Matthews, Daniel M. Tippens, Lena Tomkoetter, Elliot M. Levie, Alejandro Torres-Hernandez, Donnele Daley, George Miller. Tumor-entrained dendritic cells promote ICOS/ICOSL-dependent Th17-like responses in pancreatic adenocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3184. doi:10.1158/1538-7445.AM2015-3184