Human Lung Cancer–Derived Immunosuppressive Plasmacytoid Dendritic Cells Release IL-1α in an AIM2 Inflammasome-Dependent Manner
2015; Elsevier BV; Volume: 185; Issue: 11 Linguagem: Inglês
10.1016/j.ajpath.2015.07.009
ISSN1525-2191
AutoresRosalinda Sorrentino, Michela Terlizzi, Vincenzo Giuseppe Di Crescenzo, Ada Popolo, Michela Pecoraro, Giuseppe Perillo, Antonio Galderisi, Aldo Pinto,
Tópico(s)Galectins and Cancer Biology
ResumoPlasmacytoid dendritic cells (pDCs) highly populate lung tumor masses and are strictly correlated to bad prognosis, yet their role in lung cancer is controversial. To understand their role in lung cancer, we isolated pDCs from human samples of lung obtained from non-small cell lung cancer patients undergoing thoracic surgery. Tumor masses presented a higher percentage of pDCs than healthy tissues; pDCs were in the immunosuppressive phenotype, as determined by higher levels of CD33 and PD-L1. Despite higher HLA-A and HLA-D expression, cancerous pDCs did not exert cytotoxic activity against tumor cells but instead promoted their proliferation. In this scenario, cancerous pDCs were able to produce high levels of IL-1α. This effect was observed on the specific activation of the inflammasome absent in melanoma 2 (AIM2), which led to higher cytoplasmic calcium release responsible for calpain activation underlying IL-1α release. The blockade of type I interferon receptor and of AIM2 via the addition of LL-37 significantly reduced the release of IL-1α, which was still high after Nod-like receptor P3 inhibition via glibenclamide. More important, mitochondrial-derived reactive oxygen species sequester diminished AIM2-dependent IL-1α release. Our data demonstrate that lung tumor–associated pDCs are responsive to the activation of AIM2 that promotes calcium efflux and reactive oxygen species from mitochondria, leading to calpain activation and high levels of IL-1α, which facilitate tumor cell proliferation in the lung. Plasmacytoid dendritic cells (pDCs) highly populate lung tumor masses and are strictly correlated to bad prognosis, yet their role in lung cancer is controversial. To understand their role in lung cancer, we isolated pDCs from human samples of lung obtained from non-small cell lung cancer patients undergoing thoracic surgery. Tumor masses presented a higher percentage of pDCs than healthy tissues; pDCs were in the immunosuppressive phenotype, as determined by higher levels of CD33 and PD-L1. Despite higher HLA-A and HLA-D expression, cancerous pDCs did not exert cytotoxic activity against tumor cells but instead promoted their proliferation. In this scenario, cancerous pDCs were able to produce high levels of IL-1α. This effect was observed on the specific activation of the inflammasome absent in melanoma 2 (AIM2), which led to higher cytoplasmic calcium release responsible for calpain activation underlying IL-1α release. The blockade of type I interferon receptor and of AIM2 via the addition of LL-37 significantly reduced the release of IL-1α, which was still high after Nod-like receptor P3 inhibition via glibenclamide. More important, mitochondrial-derived reactive oxygen species sequester diminished AIM2-dependent IL-1α release. Our data demonstrate that lung tumor–associated pDCs are responsive to the activation of AIM2 that promotes calcium efflux and reactive oxygen species from mitochondria, leading to calpain activation and high levels of IL-1α, which facilitate tumor cell proliferation in the lung. In the past decade, several studies have highlighted the key role of plasmacytoid dendritic cells (pDCs) in innate immunity.1Pinto A. Rega A. Crother T.R. Sorrentino R. Plasmacytoid dendritic cells and their therapeutic activity in cancer.Oncoimmunology. 2012; 1: 726-734Crossref PubMed Scopus (31) Google Scholar, 2Vermi W. Soncini M. Melocchi L. Sozzani S. Facchetti F. Plasmacytoid dendritic cells and cancer.J Leukoc Biol. 2011; 90: 681-690Crossref PubMed Scopus (66) Google Scholar Their discovery dates back 50 years ago and, although their functional activity is nowadays being better explored, their role in specific pathologies still remains elusive. Human neoplasms, including solid tumors, such as head and neck, breast,3Treilleux I. Blay J.-Y. Bendriss-Vermare N. Ray-Coquard I. Bachelot T. Guastalla J.-P. Bremond A. Goddard S. Pin J.J. Barthelemy-Dubois C. Lebecque S. Dendritic cell infiltration and prognosis of early stage breast cancer.Clin Cancer Res. 2004; 10: 7466-7474Crossref PubMed Scopus (345) Google Scholar ovarian,4Conrad C. Gregorio J. Wang Y.-H. Ito T. Meller S. Hanabuchi S. Anderson S. Atkinson N. Ramirez P.T. Liu Y.J. Freedman R. Gilliet M. Plasmacytoid dendritic cells promote immunosuppression in ovarian cancer via ICOS costimulation of Foxp3(+) T-regulatory cells.Cancer Res. 2012; 72: 5240-5249Crossref PubMed Scopus (212) Google Scholar lung cancer,5Pinto A. Morello S. Sorrentino R. Lung cancer and Toll-like receptors.Cancer Immunol Immunother. 2011; 60: 1211-1220Crossref PubMed Scopus (61) Google Scholar and skin tumors,6Gerlini G. Urso C. Mariotti G. Di Gennaro P. Palli D. Brandani P. Salvadori A. Pimpinelli N. Reali U.M. Borgognoni L. Plasmacytoid dendritic cells represent a major dendritic cell subset in sentinel lymph nodes of melanoma patients and accumulate in metastatic nodes.Clin Immunol. 2007; 125: 184-193Crossref PubMed Scopus (64) Google Scholar are populated by nonactive/tolerogenic pDCs, associated with bad prognosis for cancer patients.7Chaput N. Conforti R. Viaud S. Spatz A. Zitvogel L. The Janus face of dendritic cells in cancer.Oncogene. 2008; 27: 5920-5931Crossref PubMed Scopus (75) Google Scholar Nonetheless, their function is still controversial. Various studies indicate that pDCs play an immunosuppressive role, and facilitate tumor progression in both animal models and humans.1Pinto A. Rega A. Crother T.R. Sorrentino R. Plasmacytoid dendritic cells and their therapeutic activity in cancer.Oncoimmunology. 2012; 1: 726-734Crossref PubMed Scopus (31) Google Scholar In contrast, others found that the presence of activated pDCs results in tumor regression in mice.1Pinto A. Rega A. Crother T.R. Sorrentino R. Plasmacytoid dendritic cells and their therapeutic activity in cancer.Oncoimmunology. 2012; 1: 726-734Crossref PubMed Scopus (31) Google Scholar Given these findings, it is clear that pDC function plays a critical role in tumor biology. pDCs are recognized as type I interferon (IFN)–producing cells.8Swiecki M. Wang Y. Vermi W. Gilfillan S. Schreiber R.D. Colonna M. Type I interferon negatively controls plasmacytoid dendritic cell numbers in vivo.J Exp Med. 2011; 208: 2367-2374Crossref PubMed Scopus (125) Google Scholar The stimulation of pDCs with Toll-like receptor (TLR) 7 and 9 ligands leads to the production of large amounts of type I IFN, underlying their potential antiviral activity.1Pinto A. Rega A. Crother T.R. Sorrentino R. Plasmacytoid dendritic cells and their therapeutic activity in cancer.Oncoimmunology. 2012; 1: 726-734Crossref PubMed Scopus (31) Google Scholar, 9McKenna K. Beignon A.-S. Bhardwaj N. Plasmacytoid dendritic cells: linking innate and adaptive immunity.J Virol. 2005; 79: 17-27Crossref PubMed Scopus (300) Google Scholar This function was also highlighted for tumor-associated pDCs (TApDCs), although pDC-derived type I IFN still needs clarification in that it can lead to both a mounting cytotoxic, antitumor activity10Swann J.B. Hayakawa Y. Zerafa N. Sheehan K.C.F. Scott B. Schreiber R.D. Hertzog P. Smyth M.J. Type I IFN contributes to NK cell homeostasis, activation, and antitumor function.J Immunol. 2007; 178: 7540-7549Crossref PubMed Scopus (231) Google Scholar and immunosuppression,11Gough D.J. Messina N.L. Clarke C.J.P. Johnstone R.W. Levy D.E. Constitutive type I interferon modulates homeostatic balance through tonic signaling.Immunity. 2012; 36: 166-174Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar which instead facilitates tumor cell immune escape. In a mouse model of melanoma, the intratumoral stimulation of pDCs with imiquimod, a TLR7 agonist, rendered these cells cytotoxic and contributed to tumor regression via IFN receptor (IFNAR) signaling.12Drobits B. Holcmann M. Amberg N. Swiecki M. Grundtner R. Hammer M. Colonna M. Sibilia M. Imiquimod clears tumors in mice independent of adaptive immunity by converting pDCs into tumor-killing effector cells.J Clin Invest. 2012; 122: 575-585Crossref PubMed Scopus (213) Google Scholar Similarly, Liu et al13Liu C. Lou Y. Lizée G. Qin H. Liu S. Rabinovich B. Kim G.J. Wang Y.H. Ye Y. Sikora A.G. Overwijk W.W. Liu Y.J. Wang G. Hwu P. Plasmacytoid dendritic cells induce NK cell-dependent, tumor antigen-specific T cell cross-priming and tumor regression in mice.J Clin Invest. 2008; 118: 1165-1175Crossref PubMed Scopus (244) Google Scholar demonstrated that the intratumoral activation of pDCs via CpG could induce natural killer cell–dependent tumor regression. In sharp contrast, stimulation of lung tumor–bearing mice with systemic CpG, a TLR9 ligand, did not lead to the same results.14Rega A. Terlizzi M. Luciano A. Forte G. Crother T.R. Arra C. Arditi M. Pinto A. Sorrentino R. Plasmacytoid dendritic cells play a key role in tumor progression in lipopolysaccharide-stimulated lung tumor-bearing mice.J Immunol. 2013; 190: 2391-2402Crossref PubMed Scopus (21) Google Scholar, 15Sorrentino R. Morello S. Luciano A. Crother T.R. Maiolino P. Bonavita E. Arra C. Adcock I.M. Arditi M. Pinto A. Plasmacytoid dendritic cells alter the antitumor activity of CpG-oligodeoxynucleotides in a mouse model of lung carcinoma.J Immunol. 2010; 185: 4641-4650Crossref PubMed Scopus (32) Google Scholar CpG-activated pDCs increased the recruitment of T-regulatory cells, and limited the inflammatory cell influx to the lung, establishing an immunosuppressive environment that favored tumor growth.1Pinto A. Rega A. Crother T.R. Sorrentino R. Plasmacytoid dendritic cells and their therapeutic activity in cancer.Oncoimmunology. 2012; 1: 726-734Crossref PubMed Scopus (31) Google Scholar, 15Sorrentino R. Morello S. Luciano A. Crother T.R. Maiolino P. Bonavita E. Arra C. Adcock I.M. Arditi M. Pinto A. Plasmacytoid dendritic cells alter the antitumor activity of CpG-oligodeoxynucleotides in a mouse model of lung carcinoma.J Immunol. 2010; 185: 4641-4650Crossref PubMed Scopus (32) Google Scholar, 16Sisirak V. Faget J. Gobert M. Goutagny N. Vey N. Treilleux I. Renaudineau S. Poyet G. Labidi-Galy S.I. Goddard-Leon S. Durand I. Le Mercier I. Bajard A. Bachelot T. Puisieux A. Puisieux I. Blay J.Y. Ménétrier-Caux C. Caux C. Bendriss-Vermare N. Impaired IFN-α production by plasmacytoid dendritic cells favors regulatory T-cell expansion that may contribute to breast cancer progression.Cancer Res. 2012; 72: 5188-5197Crossref PubMed Scopus (223) Google Scholar The same was observed in a mouse model of breast cancer in which in vivo depletion of pDCs delayed tumor growth, showing that TApDCs provided an immune-subversive environment, most likely through T-regulatory cell activation, thus favoring breast tumor progression.16Sisirak V. Faget J. Gobert M. Goutagny N. Vey N. Treilleux I. Renaudineau S. Poyet G. Labidi-Galy S.I. Goddard-Leon S. Durand I. Le Mercier I. Bajard A. Bachelot T. Puisieux A. Puisieux I. Blay J.Y. Ménétrier-Caux C. Caux C. Bendriss-Vermare N. Impaired IFN-α production by plasmacytoid dendritic cells favors regulatory T-cell expansion that may contribute to breast cancer progression.Cancer Res. 2012; 72: 5188-5197Crossref PubMed Scopus (223) Google Scholar Similarly, the high presence of pDCs contrasted doxorubicin-induced tumor cell death and, thus, regression.17Terlizzi M. Popolo A. Pinto A. Sorrentino R. Plasmacytoid dendritic cells contribute to doxorubicin-induced tumor arrest in a mouse model of pulmonary metastasis.J Immunother. 2014; 37: 214-224Crossref PubMed Scopus (3) Google Scholar In the absence of a specific stimulus, pDCs in the tumor mass have been associated with the development and maintenance of the immune-suppressive microenvironment.18Lande R. Gilliet M. Plasmacytoid dendritic cells: key players in the initiation and regulation of immune responses.Ann N Y Acad Sci. 2010; 1183: 89-103Crossref PubMed Scopus (151) Google Scholar Similar to mice, human pDCs are in their immature phenotype in tumor masses.1Pinto A. Rega A. Crother T.R. Sorrentino R. Plasmacytoid dendritic cells and their therapeutic activity in cancer.Oncoimmunology. 2012; 1: 726-734Crossref PubMed Scopus (31) Google Scholar, 7Chaput N. Conforti R. Viaud S. Spatz A. Zitvogel L. The Janus face of dendritic cells in cancer.Oncogene. 2008; 27: 5920-5931Crossref PubMed Scopus (75) Google Scholar Nonetheless, a thorough study has never been conducted on the role of these cells in human lung tumor microenvironment, which may differ from the extensively studied mouse pDCs. The phenotype of pDCs is strictly correlated to the environment they encounter.1Pinto A. Rega A. Crother T.R. Sorrentino R. Plasmacytoid dendritic cells and their therapeutic activity in cancer.Oncoimmunology. 2012; 1: 726-734Crossref PubMed Scopus (31) Google Scholar In this context, chronic inflammation and the ensuing signaling pathways in the tumor mass are relevant to decide for the fate of the infiltrated pDCs. An emerging area of investigation is the role of the inflammasome in cancer.19Terlizzi M. Casolaro V. Pinto A. Sorrentino R. Inflammasome: cancer's friend or foe?.Pharmacol Ther. 2014; 143: 24-33Crossref PubMed Scopus (65) Google Scholar The inflammasome is a multiprotein complex that comprises the assembly of Nod-like receptors (NLRs) or HIN200 family receptors, such as absent in melanoma 2 (AIM2), able to bind the adaptor apoptosis-associated speck-like protein containing a carboxyterminal CARD that induces the autocleavage of caspase-1 and the activation of IL-1–like cytokines.19Terlizzi M. Casolaro V. Pinto A. Sorrentino R. Inflammasome: cancer's friend or foe?.Pharmacol Ther. 2014; 143: 24-33Crossref PubMed Scopus (65) Google Scholar It is of great interest that high serum concentrations of proinflammatory inflammasome-related cytokines (eg, IL-1α/β and IL-18) are correlated to malignancies with a low rate of survival from time of diagnosis.20Dinarello C.A. Interleukin-1α neutralisation in patients with cancer.Lancet Oncol. 2014; 15: 552-553Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar In the tumor microenvironment, IL-1-like cytokines can be secreted by both malignant and infiltrated immune cells.21Garlanda C. Dinarello C.A. Mantovani A. The interleukin-1 family: back to the future.Immunity. 2013; 39: 1003-1018Abstract Full Text Full Text PDF PubMed Scopus (1302) Google Scholar On the basis of the notion that the inflammasome is responsible for IL-1–like cytokine release by bone marrow–derived macrophages and dendritic cells,19Terlizzi M. Casolaro V. Pinto A. Sorrentino R. Inflammasome: cancer's friend or foe?.Pharmacol Ther. 2014; 143: 24-33Crossref PubMed Scopus (65) Google Scholar the aim of our study was to understand the role of pDCs during a pathological condition, such as lung cancer. Herein, we found that human immunosuppressive TApDCs produced higher levels of IL-1α than healthy pDCs in an AIM2-dependent manner and that their activity was type I IFN and oxidative stress dependent. We used lung samples of non-small cell lung cancer patients undergoing thoracic surgery after their approval according to the Review Board of the Hospital of Salerno (Salerno, Italy). The non-small cell lung cancer subjects were 60 ± 10 years of age, and the histological status is described in Table 1. Samples from the tumor mass were defined as cancerous, whereas the healthy samples were obtained from the same patient from a distant noncancerous portion of the lung.Table 1Histological Status of the Tumor Tissues Obtained by 14 Patients with Lung CancerPatient No.Type of lung cancer1Adenocarcinoma2Adenocarcinoma3Adenocarcinoma4Squamous carcinoma5Adenocarcinoma6Adenocarcinoma7Adenocarcinoma8Squamous carcinoma9Adenocarcinoma10Adenocarcinoma11Squamous carcinoma12Adenocarcinoma13Adenocarcinoma14Squamous carcinoma Open table in a new tab pDCs were isolated from healthy and cancerous samples that were excised and digested with 1 U/mL collagenase and 20 μg/mL DNase I (Sigma Aldrich, Milan, Italy) and antibiotics. Red blood cells were lysed. pDCs were isolated using a custom negative selection in that the enrichment immunomagnetic cocktail for human pDCs (EasySep Stem Cell, Voden, Milan, Italy) was modified with the addition of anti-EpCam antibody to avoid epithelial or tumor cell contamination. Purity was checked by flow cytometry, and was routinely approximately 85% to 90% (Supplemental Figure S1A), and by means of confocal microscopy (Supplemental Figure S1B). pDCs were treated with the following: 0.1 to 10 μg/mL CpG (InvivoGen, San Diego, CA), 0.1 to 10 μg/mL imiquimod (InvivoGen), 0.1 to 10 μg/mL poly(dA:dT) (InvivoGen), 1 μmol/L glibenclamide, NLRP3 inhibitor (Sigma Aldrich, Rome, Italy), 1 μmol/L diazoxide (DZX; Sigma Aldrich, Rome, Italy), 10 μmol/L calpain inhibitor MDL 28170 (Sigma Aldrich, Rome, Italy), 10 μmol/L necrostatin-1, receptor-interacting protein kinase inhibitor (Sigma Aldrich, Rome, Italy), 10 μmol/L diphenyleneiodonium chloride (DPI), NADPH oxidase inhibitor (Sigma Aldrich, Rome, Italy), 10 μmol/L N-acetylcysteine (NAC; Sigma Aldrich, Rome, Italy), and 0.1 μg/mL LL-37 (Biolegend, San Diego, CA). Carboxyfluorescein diacetate succinimyl ester (CFSE)+ human leukemia K562 cells were cultured with pDCs (ratio, 1:2.5) for 5 to 24 hours to measure their cytotoxic activity, according to the percentage of CFSE+ propidium iodide–positive K562 cells determined by flow cytometry. In another set of experiments, we performed co-culture experiments with the human Jurkat T cell line and CpG- or phosphate-buffered saline–pulsed pDCs (ratio, 1:10 or 1:5), to test the activity of pDC antigen-presenting activity. pDCs were identified by flow cytometry (BD FacsCalibur, Milan, Italy) using the following antibodies: CD123-allophycocyanin, B220-phycoerythrin, CD19-PeCy5.5, BDCA-2 fluorescein isothiocyanate (FITC), CD33-PerCP, HLA-AI-PerCP, HLA-D–PerCP, granzyme B–FITC or allophycocyanin, and PD-L1-FITC or allophycocyanin (eBioscience, San Diego, CA). Intracellular staining for IL-1α was performed on fixed/permeabilized pDCs after the extracellular staining for CD123, B220, and CD19. Healthy and cancerous pDCs were stained for MitoSOX Mitochondrial Superoxide Indicator, as indicated in the manufacturer's guide (Life Technologies, Carlsbad, CA). Enriched pDC solution and human tissues were stained for CD123–AlexaFluor 555 (eBioscience) or BDCA-2–AlexaFluor 555 (eBioscience) and intracellular IL-1α–FITC. Similar staining was performed on human lung cancer–derived tissues. Images were observed by means of confocal microscopy (magnification, ×40; Zeiss, Jena, Germany). Caspase-1 activity was measured by means of a commercially available FAM FLICA caspase-1 assay kit (ImmunoChemistry Technologies, Bloomington, MN) and analyzed/expressed according to the absorbance (550 nm) of FLICA+ cells. IFNα, IL-18, IL-1α, and IL-1β were measured in cell-free supernatant obtained from the pDC culture by using commercially available enzyme-linked immunosorbent assays (eBioscience). Intracellular calcium concentrations were measured by using the fluorescent dye Fura 2-AM (Sigma Aldrich, Rome, Italy). pDCs (5 × 104 cells per well) were incubated at 37°C with CpG, imiquimod, and poly(dA:dT) for 1 hour. Thereafter, cells were washed and Fura 2-AM hydrolysis was allowed in calcium-free medium, as already reported.22Pecoraro M. Sorrentino R. Franceschelli S. Del Pizzo M. Pinto A. Popolo A. Doxorubicin-mediated cardiotoxicity: role of mitochondrial connexin 43.Cardiovasc Toxicol. 2015; 15: 366-376Crossref PubMed Scopus (39) Google Scholar Data were expressed as percentage of Δ increase of fluorescence ratio (F340/F380 nm) induced by 1 μmol/L ionomycin or 0.05 μmol/L carbonyl cyanide p-trifluoromethoxy-phenylhydrazone–basal fluorescence/basal fluorescence ratio (F340/F380 nm). Results are expressed as means ± SEM. A t-test or one-way analysis of variance was used to statistically evaluate differences among treatments. P < 0.05 was considered significant. Previously, we demonstrated that pDCs highly populate the lung of tumor-bearing mice participating in the establishment of the tumor immune microenvironment and progression.14Rega A. Terlizzi M. Luciano A. Forte G. Crother T.R. Arra C. Arditi M. Pinto A. Sorrentino R. Plasmacytoid dendritic cells play a key role in tumor progression in lipopolysaccharide-stimulated lung tumor-bearing mice.J Immunol. 2013; 190: 2391-2402Crossref PubMed Scopus (21) Google Scholar, 15Sorrentino R. Morello S. Luciano A. Crother T.R. Maiolino P. Bonavita E. Arra C. Adcock I.M. Arditi M. Pinto A. Plasmacytoid dendritic cells alter the antitumor activity of CpG-oligodeoxynucleotides in a mouse model of lung carcinoma.J Immunol. 2010; 185: 4641-4650Crossref PubMed Scopus (32) Google Scholar To understand the role of pDCs in humans, samples of cancerous and healthy human lung were enzymatically digested and analyzed for the presence of pDCs (identified as B220+CD19−BDCA-2+CD123+ cells) by means of flow cytometry. Human lung cancer tissues were highly populated by pDCs compared with healthy lung tissues (1.72 ± 0.36 versus 8.49 ± 1.22) (Figure 1, A and B, and Supplemental Figure S1C), confirming our previous data on mice.15Sorrentino R. Morello S. Luciano A. Crother T.R. Maiolino P. Bonavita E. Arra C. Adcock I.M. Arditi M. Pinto A. Plasmacytoid dendritic cells alter the antitumor activity of CpG-oligodeoxynucleotides in a mouse model of lung carcinoma.J Immunol. 2010; 185: 4641-4650Crossref PubMed Scopus (32) Google Scholar Interestingly, we found that healthy lung tissues had similar percentages of pDCs as healthy blood (data not shown). To understand the phenotype of pDCs, we went on by analyzing the expression of the immunosuppressive CD3323Pillai S. Netravali I.A. Cariappa A. Mattoo H. Siglecs and immune regulation.Annu Rev Immunol. 2012; 30: 357-392Crossref PubMed Scopus (246) Google Scholar and PD-L1.24Francisco L.M. Sage P.T. Sharpe A.H. The PD-1 pathway in tolerance and autoimmunity.Immunol Rev. 2010; 236: 219-242Crossref PubMed Scopus (1549) Google Scholar Lung cancer–derived pDCs presented higher levels of CD33 (Figure 1C) and PD-L1 (Figure 1D), and they also had higher levels of HLA-AI (Figure 1E), HLA-D (Figure 1F), and intracellular granzyme B (Figure 1G). Because of the high expression of HLAs, to evaluate pDC antigen-presenting activity, we performed co-culture experiments with the human Jurkat T cell line and CpG- or phosphate-buffered saline–pulsed pDCs (ratio, 1:10 or 1:5). The proliferation rate of Jurkat T cells was increased when cancerous, but not healthy, pDCs were added. However, we were not able to detect type 1 helper T-cell–like cytokines to show an antitumor activity (data not shown). According to the higher expression of granzyme B, pDCs were co-cultured with the K562 cell line, previously stained with CFSE, to measure pDC cytotoxic activity. The addition of lung cancer–derived (cancerous) pDCs to CFSE+ K562 cells significantly reduced the percentage of CFSEhigh propidium iodide–positive cells (Figure 2A), but, instead, a higher percentage was registered for CFSE+ propidium iodide–negative K562 cells (Figure 2B), compared with healthy pDCs, suggesting that K562 cells were induced to cell proliferation. These data highlight that cancerous immunosuppressive pDCs facilitate tumor cell proliferation in human lung cancer. pDCs are highly specialized at sensing nucleic acids via the intracellular pattern recognition receptors TLR7 and TLR9, which lead to the release of high levels of type I IFN.1Pinto A. Rega A. Crother T.R. Sorrentino R. Plasmacytoid dendritic cells and their therapeutic activity in cancer.Oncoimmunology. 2012; 1: 726-734Crossref PubMed Scopus (31) Google Scholar Hence, pDCs are recognized as the professional IFN-producing cells. The stimulation of healthy pDCs with 0.1 μg/mL CpG and 1 μg/mL imiquimod significantly increased the production of IFNα (Figure 3, A and B). Instead, cancerous pDCs were not able to release higher levels of IFNα under CpG (Figure 3A) and imiquimod stimulation (Figure 3B), although the basal levels of IFNα in these cells were significantly higher than the basal levels of healthy pDCs (Figure 3, A and B). Because a recent study found that type I IFN negatively controls pDC turnover in that an overproduction of type I IFNs can lead to the death of pDCs during steady-state conditions and viral infections,8Swiecki M. Wang Y. Vermi W. Gilfillan S. Schreiber R.D. Colonna M. Type I interferon negatively controls plasmacytoid dendritic cell numbers in vivo.J Exp Med. 2011; 208: 2367-2374Crossref PubMed Scopus (125) Google Scholar we measured lactate dehydrogenase (LDH) release. Both healthy and cancerous pDCs were not dying cells because the levels of LDH were not as high as the positive control (Figure 3C). On the basis of the notion that IL-1–like cytokines highly populate tumor masses,20Dinarello C.A. Interleukin-1α neutralisation in patients with cancer.Lancet Oncol. 2014; 15: 552-553Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar we kept on our study by investigating whether cancerous pDCs were able to release IL-1α and IL-1β under inflammasome activation. We first measured the expression of AIM2 and NLRP3 by means of flow cytometry. The levels of AIM2 (Figure 3, D and E) and NLRP3 (Figure 3, E and F) were significantly increased in cancerous pDCs compared with healthy cells. Interestingly, the stimulation of healthy and cancerous pDCs with poly(dA:dT), an AIM2 ligand, did not increase the levels of IL-1β (Figure 4A), whereas the stimulation of TApDCs with a well-known NLRP3 activator, ATP, increased IL-1β levels compared with healthy pDCs (healthy pDCs: control versus ATP stimulated, 68.47 ± 11.18 versus 47.7 ± 11.37; TApDCs: control versus ATP stimulated, 75.88 ± 12.66 versus 142.9 ± 13.3; data not shown). Interestingly, both healthy and cancerous pDCs showed higher levels of FAM-FLICA absorbance, confirming the activation of caspase-1 after poly(dA:dT) addition (Figure 4B), although these cells were not LDH-releasing cells (Figure 4C). In sharp contrast, poly(dA:dT) robustly increased the release of IL-1α from cancerous pDCs compared with healthy pDCs (Figure 4D). To confirm that pDCs were the source of IL-1α in our experimental conditions, we performed both confocal microscopy (Supplemental Figure S1, B and C) and flow cytometry analyses (Figure 4, E and F). Non-stimulated (control) lung cancer–derived pDCs (CD123+ cells) had intracellular stores of IL-1α (Supplemental Figure S1B). Concomitantly, the stimulation of lung cancer–derived pDCs with phosphate-buffered saline or poly(dA:dT) for 5 hours decreased the levels of the intracellular IL-1α (Figure 4, E and F), although not in a statistically significant manner (P = 0.0844), confirming the release of the cytokine into the supernatant, as shown in Figure 4D. Moreover, to avoid in vitro limitations, we also stained lung cancer tissues for intracellular IL-1α (FITC). CD123+ cells were in the tumor microenvironment, surrounding the tumor mass, highlighted by the DAPI staining (Supplemental Figure S1C). CD123+ cells were IL-1α–FITC positive, confirming that pDCs are another source of IL-1α in the lung tumor microenvironment (Supplemental Figure S1C). To further evaluate the role of the AIM2–caspase-1 inflammasome in cancerous pDCs, we treated the cells with y-Vad, a well-known caspase-1 inhibitor. The inhibition of caspase-1 significantly reduced the release of IL-1α from cancerous pDCs after poly(dA:dT) stimulation (Figure 4G). The same effect was observed with an aspecific caspase inhibitor, z-VAD (Figure 4H). To investigate the molecular mechanism underlying AIM2 activation, we went on by blocking type I IFNAR by means of a monoclonal antibody. The blockade of IFNAR reduced the release of IL-1α after poly(dA:dT) treatment (Figure 4I), implying that type I IFN is involved in the induction of IL-1α after AIM2 activation. Moreover, to confirm the involvement of AIM2, we observed that the addition of LL-37, an AIM2 inhibitor,25Rathinam V.A.K. Jiang Z. Waggoner S.N. Sharma S. Cole L.E. Waggoner L. Vanaja S.K. Monks B.G. Ganesan S. Latz E. Hornung V. Vogel S.N. Szomolanyi-Tsuda E. Fitzgerald K.A. The AIM2 inflammasome is essential for host defense against cytosolic bacteria and DNA viruses.Nat Immunol. 2010; 11: 395-402Crossref PubMed Scopus (960) Google Scholar significantly reduced the levels of IL-1α after poly(dA:dT) addition (Figure 4J). To confirm that poly(dA:dT) activity was solely AIM2 dependent, we treated TApDCs with a well-known inhibitor of NLRP3,26Lamkanfi M. Mueller J.L. Vitari A.C. Misaghi S. Fedorova A. Deshayes K. Lee W.P. Hoffman H.M. Dixit V.M. Glyburide inhibits the Cryopyrin/Nalp3 inflammasome.J Cell Biol. 2009; 187: 61-70Crossref PubMed Scopus (590) Google Scholar glibenclamide, and a K/ATP channel opener, DZX. The inhibition of NLRP3 by means of glibenclamide did not alter poly(dA:dT)-induced IL-1α release (Figure 5A). Similarly, the activation of NLRP3 by means of DZX did not modify poly(dA:dT)-induced IL-1α release (Figure 5B), implying that AIM2 was responsible for IL-1α release. Cell viability was not altered after the addition of glibenclamide and DZX (data not shown). England et al27England H. Summersgill H.R. Edye M.E. Rothwell N.J. Brough D. Release of interleukin-1α or interleukin-1β depends on mechanism of cell death.J Biol Chem. 2014; 289: 15942-15950Crossref PubMed Scopus (102) Google Scholar described that IL-1α is not a substrate for caspase-1. Similarly, Gross et al28Gross O. Yazdi A.S. Thomas C.J. Masin M. Heinz L.X. Guarda G. Quadroni M. Drexler S.K. Tschopp J. Inflammasome activators induce interleukin-1α secretion via distinct pathways with differential requirement for the protease function of caspase-1.Immunity. 2012; 36: 388-400Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar showed that IL-1α is not universall
Referência(s)