Faculty Bibliography

Con A hepatitis is regarded as a T cell-mediated model of acute liver injury. Mincle is a C-type lectin receptor that is critical in the immune response to mycobacteria and fungi but does not have a well-defined role in preclinical models of non-pathogen-mediated inflammation. Because Mincle can ligate the cell death ligand SAP130, we postulated that Mincle signaling drives intrahepatic inflammation and liver injury in Con A hepatitis. Acute liver injury was assessed in the murine Con A hepatitis model using C57BL/6, Mincle-/-, and Dectin-1-/- mice. The role of C/EBPbeta and hypoxia-inducible factor-1alpha (HIF-1alpha) signaling was assessed using selective inhibitors. We found that Mincle was highly expressed in hepatic innate inflammatory cells and endothelial cells in both mice and humans. Furthermore, sterile Mincle ligands and Mincle signaling intermediates were increased in the murine liver in Con A hepatitis. Most significantly, Mincle deletion or blockade protected against Con A hepatitis, whereas Mincle ligation exacerbated disease. Bone marrow chimeric and adoptive transfer experiments suggested that Mincle signaling in infiltrating myeloid cells dictates disease phenotype. Conversely, signaling via other C-type lectin receptors did not alter disease course. Mechanistically, we found that Mincle blockade decreased the NF-kappabeta-related signaling intermediates C/EBPbeta and HIF-1alpha, both of which are necessary in macrophage-mediated inflammatory responses. Accordingly, Mincle deletion lowered production of nitrites in Con A hepatitis and inhibition of both C/EBPbeta and HIF-1alpha reduced the severity of liver disease. Our work implicates a novel innate immune driver of Con A hepatitis and, more broadly, suggests a potential role for Mincle in diseases governed by sterile inflammation.

Inflammation is paramount in pancreatic oncogenesis. We identified a uniquely activated gammadeltaT cell population, which constituted approximately 40% of tumor-infiltrating T cells in human pancreatic ductal adenocarcinoma (PDA). Recruitment and activation of gammadeltaT cells was contingent on diverse chemokine signals. Deletion, depletion, or blockade of gammadeltaT cell recruitment was protective against PDA and resulted in increased infiltration, activation, and Th1 polarization of alphabetaT cells. Although alphabetaT cells were dispensable to outcome in PDA, they became indispensable mediators of tumor protection upon gammadeltaT cell ablation. PDA-infiltrating gammadeltaT cells expressed high levels of exhaustion ligands and thereby negated adaptive anti-tumor immunity. Blockade of PD-L1 in gammadeltaT cells enhanced CD4+ and CD8+ T cell infiltration and immunogenicity and induced tumor protection suggesting that gammadeltaT cells are critical sources of immune-suppressive checkpoint ligands in PDA. We describe gammadeltaT cells as central regulators of effector T cell activation in cancer via novel cross-talk.

Regulation of Toll-like receptor responses is critical for limiting tissue injury and autoimmunity in both sepsis and sterile inflammation. We found that Mincle, a C-type lectin receptor, regulates proinflammatory Toll-like receptor 4 signaling. Specifically, Mincle ligation diminishes Toll-like receptor 4-mediated inflammation, whereas Mincle deletion or knockdown results in marked hyperresponsiveness to lipopolysaccharide in vitro, as well as overwhelming lipopolysaccharide-mediated inflammation in vivo. Mechanistically, Mincle deletion does not up-regulate Toll-like receptor 4 expression or reduce interleukin 10 production after Toll-like receptor 4 ligation; however, Mincle deletion decreases production of the p38 mitogen-activated protein kinase-dependent inhibitory intermediate suppressor of cytokine signaling 1, A20, and ABIN3 and increases expression of the Toll-like receptor 4 coreceptor CD14. Blockade of CD14 mitigates the increased sensitivity of Mincle-/- leukocytes to Toll-like receptor 4 ligation. Collectively, we describe a major role for Mincle in suppressing Toll-like receptor 4 responses and implicate its importance in nonmycobacterial models of inflammation.

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 outcome, 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 pre-invasive foci. METHODS: We investigated the effects of radiation in p48Cre;LSL-KrasG12D (KC) and p48Cre;LSLKrasG12D;LSL-Trp53R172H (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 2-12 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 invasive and pre-invasive 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. An 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 exposure causes macrophages in PDAs of mice to acquire an immune-suppressive phenotype and reduce T-cell mediated anti-tumor responses. Agents that block MCSF prevent this effect, allowing radiation to have increased efficacy in slowing tumor growth.

Assumptions that liver immune cells and immunosuppressive pathways are similar to their counterparts in other spaces have led to gaps in our understanding of intrahepatic neoplasm aggressiveness. Myeloid-derived suppressor cells (MDSCs) are potent inhibitors of antitumor immunity and pose a major obstacle to solid tumor treatment. Liver MDSCs (L-MDSCs) associated with liver metastases (LM) are particularly problematic by contributing to intrahepatic immunosuppression that promotes tumor progression. L-MDSCs have been reported to expand in response to granulocyte-macrophages colony-stimulating factor (GM-CSF) and suppress antitumor immunity in LM. To extend these findings, we examined mechanisms of intrahepatic immunosuppression exploited by L-MDSCs. We found that the majority of L-MDSCs co-expressed GM-CSF receptor (GM-CSF-R), indoleamine 2,3-dioxygenase (IDO) and programmed death ligand 1 (PD-L1), while demonstrating high levels of signal transducer and activator of transcription factor 3 (STAT3) activation. GM-CSF-secreting tumor cells induced STAT3 phosphorylation in L-MDSCs in addition to expression of IDO and PD-L1. GM-CSF or GM-CSF-R blockade markedly reduced L-MDSC IDO and PD-L1 expression, implicating tumor-derived GM-CSF in supporting L-MDSC-immunoinhibitory molecule expression. Small-molecule inhibitors of Janus-activated kinase 2 (JAK2) and STAT3 also dramatically diminished IDO and PD-L1 expression in L-MDSCs. We determined that STAT3 exerts transcriptional control over L-MDSC IDO and PD-L1 expression by binding to the IDO1 and PD-L1 promoters. Our data suggest that the GM-CSF/JAK2/STAT3 axis in L-MDSCs drives immunosuppression in a model of LM and blockade of this pathway may enable rescue of intrahepatic antitumor immunity.Cancer Gene Therapy advance online publication, 20 May 2016; doi:10.1038/cgt.2016.19.

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.

Introduction: Disruption of apoptosis is a basic modality cancer cells exploit for survival. However, the role of programmed necrosis in the life cycle of pancreatic ductal adenocarcinoma (PDA) is uncertain. Here we report that the principal components of the necrosome, RIP1 and RIP3, are highly expressed in pancreatic ductal adenocarcinoma (PDA) and are further upregulated by chemotherapeutics. Methods: We evaluated the effects of deletion orblockade of the necroptosis pathway in pancreatic cancer on tumor size and survival, peritumoral fibroinflammation, and epithelial transformation. We utilized p48Cre;KrasG12D(KC) mice as our murine PDA oncogenesis model and crossed KC with RIP3-/- and Mincle-/- mice to create a knockout pancreatic cancer mouse model. Alternatively, we challenged wildtype mice with orthopic injection of the cancer cell line FC1242 into the pancreas. Components of the necroptosis pathway and the immune infiltrate within the pancreatic TME were assessed and characterized using immunohistochemistry, flow cytometry and western blotting in human and murine tissue. Results: Blockade of the necrosome in vitro promoted cancer cell proliferation and induced an aggressive oncogenic phenotype in transformed pancreatic epithelial cells. Conversely, in vivo RIP3 deletion or RIP1 inhibition was protective against oncogenic progression and was associated with the development of a highly immunogenic myeloid and T cell phenotype within the tumor microenvironment (TME). The immunesuppressive infiltrate associated with intact RIP1/RIP3 signaling was contingent on necroptosisinduced CXCL1 expression whereas CXCL1 blockade was protective against PDA. Moreover, we found that the necroptotic byproduct SAP130 was highly prevalent in PDA and Mincle - its cognate receptor - was upregulated in tumorinfiltrating myeloid cells. Mincle ligation powerfully promoted oncogenesis whereas Mincle deletion was protective against tumorigenesis and phenocopied the immunogenic reprogramming of the TME characteristic of RIP3 deletion. Conclusion: Our work describes a novel RIP1/RIP3-Mincle axis as a critical regulator of peritumoral immune suppression and PDA progression