Myeloid-derived suppressor cells (MDSC) accumulate in the spleen and tumor bed during tumor growth. They contribute to the immune tolerance of cancer notably by inhibiting the function of CD8 (+) T cells. Thus, their elimination may hamper tumor growth by enhancing antitumor T-cell functions. We have previously reported that some anticancer agents relied on T cell-dependent anticancer responses to achieve maximal efficacy. However, the effect of anticancer agents on MDSC has remained largely unexplored. In this study, we observed that gemcitabine and 5-fluorouracil (5FU) were selectively cytotoxic on MDSC. In vivo, the treatment of tumor-bearing mice with 5FU led to a major decrease in the number of MDSC in the spleens and tumor beds of animals whereas no significant effect on T cells, natural killer cells, dendritic cells, or B cells was noted. Interestingly, 5FU showed a stronger efficacy over gemcitabine to deplete MDSC and selectively induced MDSC apoptotic cell death in vitro and in vivo. The elimination of MDSC by 5FU increased IFN-γ production by tumor-specific CD8(+) T cells infiltrating the tumor and promoted T cell-dependent antitumor responses in vivo. Altogether, these findings suggest that the antitumor effect of 5FU is mediated, at least in part, by its selective cytotoxic action on MDSC. Cancer Res; 70(8); 3052-61. ©2010 AACR.

Chemotherapeutic agents are widely used for cancer treatment. In addition to their direct cytotoxic effects, these agents harness the host's immune system, which contributes to their antitumor activity. Here we show that two clinically used chemotherapeutic agents, gemcitabine (Gem) and 5-fluorouracil (5FU), activate the NOD-like receptor family, pyrin domain containing-3 protein (Nlrp3)-dependent caspase-1 activation complex (termed the inflammasome) in myeloid-derived suppressor cells (MDSCs), leading to production of interleukin-1β (IL-1β), which curtails anticancer immunity. Chemotherapy-triggered IL-1β secretion relied on lysosomal permeabilization and the release of cathepsin B, which bound to Nlrp3 and drove caspase-1 activation. MDSC-derived IL-1β induced secretion of IL-17 by CD4(+) T cells, which blunted the anticancer efficacy of the chemotherapy. Accordingly, Gem and 5FU exerted higher antitumor effects when tumors were established in Nlrp3(-/-) or Casp1(-/-) mice or wild-type mice treated with interleukin-1 receptor antagonist (IL-1Ra). Altogether, these results identify how activation of the Nlrp3 inflammasome in MDSCs by 5FU and Gem limits the antitumor efficacy of these chemotherapeutic agents.

Although Th17 cells are known to promote tissue inflammation and autoimmunity, their role during cancer progression remains elusive. Here, we showed that in vitro Th17 cells generated with the cytokines IL-6 and TGF-β expressed CD39 and CD73 ectonucleotidases, leading to adenosine release and the subsequent suppression of CD4(+) and CD8(+) T cell effector functions. The IL-6-mediated activation of the transcription factor Stat3 and the TGF-β-driven downregulation of Gfi-1 transcription factor were both essential for the expression of ectonucleotidases during Th17 cell differentiation. Stat3 supported whereas Gfi-1 repressed CD39 and CD73 expression by binding to their promoters. Accordingly, Th17 cells differentiated with IL-1β, IL-6, and IL-23 but without TGF-β did not express ectonucleotidases and were not immunosuppressive. Finally, adoptive transfer of Th17 cells induced by TGF-β and IL-6 promoted tumor growth in a CD39-dependent manner. Thus, ectonucleotidase expression supports the immunosuppressive fate of Th17 cells in cancer.

The receptor NLRP3 is involved in the formation of the NLRP3 inflammasome that activates caspase-1 and mediates the release of interleukin 1β (IL-1β) and IL-18. Whether NLRP3 can shape immunological function independently of inflammasomes is unclear. We found that NLRP3 expression in CD4(+) T cells specifically supported a T helper type 2 (TH2) transcriptional program in a cell-intrinsic manner. NLRP3, but not the inflammasome adaptor ASC or caspase-1, positively regulated a TH2 program. In TH2 cells, NLRP3 bound the Il4 promoter and transactivated it in conjunction with the transcription factor IRF4. Nlrp3-deficient TH2 cells supported melanoma tumor growth in an IL-4-dependent manner and also promoted asthma-like symptoms. Our results demonstrate the ability of NLRP3 to act as a key transcription factor in TH2 differentiation.

Host immunity controls the development of colorectal cancer, and chemotherapy used to treat colorectal cancer is likely to recruit the host immune system at some level. Athough preclinical studies have argued that colorectal cancer drugs, such as 5-fluorouracil (5-FU) and oxaliplatin, exert such effects, their combination as employed in the oncology clinic has not been evaluated. Here, we report the results of prospective immunomonitoring of 25 metastatic colorectal cancer (mCRC) patients treated with a first-line combination regimen of 5-FU, oxaliplatin, and bevacizumab (FOLFOX-bevacizumab), as compared with 20 healthy volunteers. Before this therapy was initiated, T regulatory cells (Treg), Th17, and granulocytic myeloid-derived suppressor cells (gMDSC) were increased significantly in mCRC, but only a high level of gMDSC was associated with a poor prognosis. Chemotherapy modulated the Treg/Th17 balance by decreasing Treg and increasing Th17 cell frequency by 15 days after the start of treatment. Increased Th17 frequency was associated with a poor prognosis. FOLFOX-bevacizumab treatment elicited a decrease in gMDSC in 15 of 25 patients and was associated with a better survival outcome. Notably, the gMDSCs that expressed high levels of PD-L1, CD39, and CD73 exerted a robust immunosuppressive activity, relative to other myeloid cells present in blood, which could be reversed by blocking the CD39/CD73 and PD-1/PD-L1 axes. Our work underscores the critical prognostic impact of early modifications in Th17 and gMDSC frequency in mCRC. Furthermore, it provides a clinical rationale to combine FOLFOX-bevacizumab chemotherapy with inhibitors of ATP ectonucleotidases and/or anti-PD-1/PD-L1 antibodies to more effectively treat this disease. Cancer Res; 76(18); 5241-52. ©2016 AACR.

Liver X receptors (LXRs) have been proposed to have some anticancer properties, through molecular mechanisms that remain elusive. Here we report for the first time that LXR ligands induce caspase-1-dependent cell death of colon cancer cells. Caspase-1 activation requires Nod-like-receptor pyrin domain containing 3 (NLRP3) inflammasome and ATP-mediated P2 Â 7 receptor activation. Surprisingly, LXRb is mainly located in the cytoplasm and has a non-genomic role by interacting with pannexin 1 leading to ATP secretion. Finally, LXR ligands have an antitumoral effect in a mouse colon cancer model, dependent on the presence of LXRb, pannexin 1, NLRP3 and caspase-1 within the tumor cells. Our results demonstrate that LXRb, through pannexin 1 interaction, can specifically induce caspase-1-dependent colon cancer cell death by pyroptosis.

The mechanisms leading to NOD-leucine rich repeat and pyrin containing protein 3 (NLRP3) inflammasome activation are still debated. It is well established that oligomerized NLRP3 interacts with apoptosis associated Speck-like protein containing a CARD domain (ASC) which polymerizes into filaments recruiting procaspase-1, leading to its activation. However, pathways triggering NLRP3 activation, such as potassium efflux, ROS production or lysosomal permeabilization, can be required or not, depending on the activators used. Here we proposed to evaluate the importance of Cathepsin B on NLRP3 inflammasome assembly and activation. Using Cathepsin B −/− BMDMs (Bone Marrow-Derived Macrophages), we first show that Cathepsin B is required for caspase-1 activation, IL-1β production and ASC speck formation, upon treatment with different types of NLRP3 activators, i.e., ATP, nigericin or crystals. Moreover, in these conditions, Cathepsin B interacts with NLRP3 at the endoplasmic reticulum (ER) level. To conclude, different NLRP3 activators lead to Cathepsin B interaction with NLRP3 at the ER level and to subsequent caspase-1 activation.

Key Words: LXR Ⅲ RAR Ⅲ macrophages Ⅲ phagocytosis Ⅲ transglutaminase L iver X receptors (LXRs) ␣ and ␤ are nuclear receptors activated by oxysterols. 1,2 They control the expression of genes involved in lipid metabolism and inflammation. 3 LXR␣ is expressed primarily in liver, intestine, adipose tissue, and macrophages, whereas LXR␤ is widely expressed. 1,2 Because LXRs, especially LXR␣, are present at high levels in macrophages, the effects of LXR activation or repression on macrophage functions have been widely investigated. 3,4 Strong evidence suggests that LXRs are master regulators of cholesterol metabolism in these cells. LXR activation improves cholesterol homeostasis and stimulates cholesterol efflux through the coordinated regulation of apolipoprotein E and cholesterol transporters such as ABCA1 (ATP-binding cassette subfamily A member 1) and ABCG1 (ATP-binding cassette subfamily G member 1). [5][6][7] LXRs agonists also modulate the functions of macrophages in innate immunity and inflammation. Synthetic LXR agonists attenuate the inflammatory response of murine macrophages exposed to lipopolysaccharides 8 and increase macrophage survival in response to bacterial infection. 9 In humans, short-term exposure of macrophages to LXR agonists also reduces the inflammatory effects of lipopolysaccharides whereas longterm exposure increases macrophage response to lipopolysaccharides through Toll-like receptor 4 upregulation. 10 Macrophages come from the differentiation of peripheral blood monocytes, which are produced in the bone marrow by differentiation of stem cells, circulate in the blood Ϸ1 to 3 days, and then are recruited in tissues where they undergo differentiation. The role of LXRs has been less studied in monocytes than in macrophages but synthetic LXR agonists MethodsAn expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org. Human peripheral blood monocytes were obtained from healthy donors, purified with the Monocyte Isolation Kit II (Miltenyi Biotec), and differentiated into macrophages with macrophagecolony stimulating factor. Mouse bone marrow-derived macrophages were collected from wild-type (WT) or LXR␣/␤ knockout (KO) mouse tibias and femurs. Microarray procedures were performed by Miltenyi Biotec; quantitative polymerase chain reaction (PCR) was performed by using a SYBR Green Real-time PCR kit (Invitrogen) on a LightCycler 2.0 detection system (Roche Diagnostics); chromatin immunoprecipitation was performed by using a ChIP-IT kit (Active Motif); and luciferase reporter assays were performed with the Dual Glo Luciferase Assay System (Promega). All-trans retinoic acid levels in human atherosclerotic plaques were determined by liquid chromatography-mass spectrometry (LC-MS). Phagocytosis was measured in macrophages incubated with 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester-stained apoptotic NB4 cells and analyzed with a LSRII flow cytometer (Becton Dickinson). Data are meansϮSD and Mann-Whitney U test was used to determine their sig...