Autophagy is a key regulator of cellular homeostasis that can be activated by pathogen-associated molecules and recently has been shown to influence IL-1␤ secretion by macrophages. However, the mechanisms behind this are unclear. Here, we describe a novel role for autophagy in regulating the production of IL-1␤ in antigen-presenting cells. After treatment of macrophages with Toll-like receptor ligands, pro-IL-1␤ was specifically sequestered into autophagosomes, whereas further activation of autophagy with rapamycin induced the degradation of pro-IL-1␤ and blocked secretion of the mature cytokine. Inhibition of autophagy promoted the processing and secretion of IL-1␤ by antigen-presenting cells in an NLRP3-and TRIF-dependent manner. This effect was reduced by inhibition of reactive oxygen species but was independent of NOX2. Induction of autophagy in mice in vivo with rapamycin reduced serum levels of IL-1␤ in response to challenge with LPS. These data demonstrate that autophagy controls the production of IL-1␤ through at least two separate mechanisms: by targeting pro-IL-1␤ for lysosomal degradation and by regulating activation of the NLRP3 inflammasome.IL-1␤ is an important proinflammatory cytokine, released at sites of infection or injury, that regulates diverse physiological responses, including cellular recruitment, appetite, sleep, and body temperature (1). IL-1␤ is first produced as a proform in response to inflammatory stimuli, such as TLR ligands. This inactive precursor is cleaved into the bioactive (p17) molecule by caspase 1, following the activation of an inflammasome (2).Inflammasomes are molecular scaffolds that trigger the activation of caspase 1 and subsequent maturation of IL-1␤ and IL-18. Typically, inflammasomes are formed from at least one member of the cytosolic innate immune sensor family, the NOD-like receptors (NLRs), including NLRP1, NLRP3, and NLRC4, coupled with the adaptor apoptosis-associated specklike protein containing a caspase recruitment domain (ASC or PYCARD) and caspase 1 (2). The NLRP3 inflammasome is the best characterized to date and is activated by a number of endogenous and exogenous signals.Most studies in vitro employ TLR ligands, particularly LPS, to induce pro-IL-1␤ formation, but in many cases, this is not enough to stimulate inflammasome activation and secretion of the mature cytokine. Instead, a second signal is commonly required, and this can come from a number of endogenous and exogenous sources, including ATP and particulates, including uric acid crystals, amyloid-␤, silica, asbestos, synthetic microparticles, and alum (3-8). Extracellular ATP triggers the P2X7 ATP-gated ion channel, leading to K ϩ efflux and induces recruitment of the pannexin-1 membrane pore (9). This may then allow extracellular NLRP3 agonists to enter the cell and activate inflammasome assembly (9). Particulates have been proposed to act through one of two mechanisms. Uptake of particulates by phagocytes may lead to lysosomal damage and release of lysosomal products into the cytosol, which a...

Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that infects alveolar macrophages following aerosol transmission. Lung macrophages provide a critical intracellular niche that is required for Mtb to establish infection in the human host. This parasitic relationship is made possible by the capacity of Mtb to block phagosome maturation following entry into the host macrophage, creating an environment that supports bacillary replication. Apoptosis is increasingly understood to play a role in host defense against intracellular pathogens including viruses, fungi, protozoa and bacteria. In the last 15 years an understanding of the role that macrophage apoptosis plays in TB has begun to emerge. Here we review the history and current state of the art of this topic and we offer a model of the macrophage-pathogen interaction that takes into the account the complexities of programmed cell death and the relationship between various death signaling pathways and host defense in TB.

Sayitoglu et al. Boosting NK Cell-Mediated Sarcoma Targeting cells was observed against the majority of tumor cell lines tested. In conclusion, DNAM-1 or NKG2D over-expression elicited a dynamic increase in NK cell degranulation against all sarcoma explants and cancer cell lines tested, including those that failed to induce a notable response in WT NK-92 cells. These results support the broad therapeutic potential of DNAM-1 + or NKG2D + GM NK-92 cells and GM human NK cells for the treatment of sarcomas and other malignancies.

To characterize the metabolic role of peroxisomes in yeast cells under physiological conditions, we performed a comprehensive meta-analysis of published microarray data. Previous studies of yeast peroxisomes have mainly been focused on the function of peroxisomes under extreme conditions, such as growth on oleate or methanol as the sole carbon source, and may therefore not be representative of the normal physiological role of yeast peroxisomes. Surprisingly, our analysis of the microarray data reveals that the only pathway responding to peroxisome deficiency in mid-log phase is lysine biosynthesis, whereas classical peroxisomal pathways such as beta-oxidation are unaffected. We show that the upregulation of lysine biosynthesis genes in peroxisome-deficient yeasts shares many characteristics with the physiological response to lysine starvation. We provide data that suggest that this is the result of a "pathological" stimulation of the Lys14p transcriptional activator by the pathway intermediate aminoadipate semialdehyde. Mistargeting of the peroxisomal lysine pathway to the cytosol increases the active concentration of aminoadipate semialdehyde, which is no longer contained in the peroxisome and can now activate Lys14p at much lower levels than in wild-type yeasts. This is the first well-documented example of pathway misregulation in response to peroxisome deficiency and will be useful in understanding the phenotypic details of human peroxisome-deficient patients (Zellweger syndrome).Peroxisomes are single-membrane-bounded organelles present in most eukaryotic cells, with the exception of Plasmodium, Giardia, Trichomonas, Entamoeba, and related species. Peroxisomes typically contain the enzymes of fatty acid betaoxidation and catalase, which converts the hydrogen peroxide formed by fatty acyl-coenzyme A (CoA) oxidase to water and oxygen. Several other oxidases, e.g., monoamine oxidase, urate oxidase, hydroxyacid oxidase, methanol oxidase, and acetylspermidine oxidase, are also present in peroxisomes of some species and benefit from the same detoxification mechanism. In addition to these oxidative reactions, peroxisomes may contain many other biosynthetic pathways. Enzymes of these supplemental pathways are usually absent or localized in nonperoxisomal compartments in most eukaryotes, and their peroxisomal localization is a specialization of relatively few species. Examples of facultative peroxisomal pathways are glycolysis and purine salvage (in the peroxisomes of kinetoplastids, such as Trypanosoma and Leishmania [23]), isoprenoid biosynthesis (in vertebrates [1]), ether phospholipid biosynthesis (in kinetoplastids and animals [15,33]), and the glyoxylate cycle, which channels the products of beta-oxidation into gluconeogenesis in plants, yeasts, and Caenorhabditis elegans (7).Peroxisome-deficient mutants of most organisms are viable (with the possible exception of kinetoplastids), but a lack of functional peroxisomes can lead to severe pathologies in multicellular organisms (3,5,8,16,20,24). In all organisms ex...

A high intracellular bacillary load of Mycobacterium tuberculosis in macrophages induces an atypical lysosomal cell death with early features of apoptosis that progress to necrosis within hours. Unlike classical apoptosis, this cell death mode does not appear to diminish M. tuberculosis viability. We previously reported that culturing heavily infected macrophages with naïve macrophages produced an antimicrobial effect, but only if naïve macrophages were added during the pre-necrotic phase of M. tuberculosis-induced cell death. In the present study we investigated the mechanism of antimicrobial activity in co-cultures, anticipating that efferocytosis of bacilli in apoptotic bodies would be required. Confocal microscopy revealed frustrated phagocytosis of M. tuberculosis-infected macrophages with no evidence that significant numbers of bacilli were transferred to the naïve macrophages. The antimicrobial effect of naïve macrophages was retained when they were separated from infected macrophages in transwells, and conditioned co-culture supernatants transferred antimicrobial activity to cultures of infected macrophages alone. Antimicrobial activity in macrophage co-cultures was abrogated when the naïve population was deficient in IL-1 receptor or when the infected population was deficient in inducible nitric oxide synthase. The participation of nitric oxide suggested a conventional antimicrobial mechanism requiring delivery of bacilli to a late endosomal compartment. Using macrophages expressing GFP-LC3 we observed the induction of autophagy specifically by a high intracellular load of M. tuberculosis. Bacilli were identified in LC3-positive compartments and LC3-positive compartments were confirmed to be acidified and LAMP1 positive. Thus, the antimicrobial effect of naïve macrophages acting on M. tuberculosis in heavily-infected macrophages is contact-independent. Interleukin-1 provides an afferent signal that induces an as yet unidentified small molecule which promotes nitric oxide-dependent antimicrobial activity against bacilli in autolysosomes of heavily infected macrophages. This cooperative, innate antimicrobial interaction may limit the maximal growth rate of M. tuberculosis prior to the expression of adaptive immunity in pulmonary tuberculosis.

Sarcomas are rare malignancies of mesenchymal origin for which current treatment options are very limited. Due to the vast heterogeneity of the various subtypes, the development of new targeted therapies requires a customized approach. Checkpoint blockade therapies are a promising alternative that still have not been systematically investigated for sarcoma patients. Thus, utilizing the infrastructure of the Cell Therapy Core Facility (CTC) at Nova Southeastern University (NSU), we developed a clinic-to-bench pipeline that enables the processing of fresh tumor material after surgical excision to generate primary sarcoma cell lines for biobanking and research purposes. Using this pipeline, we have extensively characterized mRNA, miRNA and cell surface expression profiles from more than 40 sarcoma cell lines to date, as well as validated their tumorigenicity by assessing expression of sarcoma/cancer associated genes such as Birc5, Birc2, Bcl2, Ewsr1-fli1, Syt/Ssx and Pcna. Moreover, we assessed the expression of immunomodulatory molecules that may suppress antitumor responses, such as PD-L1, PD-L2, OX40L and CD40L, using multicolor flow cytometry. Detailed characterization of the immune profile of 7 bone-related and 7 soft tissue sarcoma cell lines, assessed after 12 weeks of in vitro serial passaging, revealed a common signature in the cell surface expression of proliferative marker PCNA, and lymphocyte ligands CD112 and CD115. Moreover, immunoprofiling of the PCNA/CD112/CD155+ sarcoma cell lines (n=14) demonstrated persistent expression of PD-L2 in all cell lines and the potential applications of currently clinically approved anti-PD-1 checkpoint inhibitors for sarcoma treatment. Importantly, the conserved expression of these proteins enabled the distinction of sarcoma tumor cells from other cells in the fresh primary tumor mix directly after isolation. Thus, we next performed a comparative analysis of the immune profile of 6 freshly isolated sarcoma tumors and their respective cell lines. Comparison of the surface expression profiles of the generated cell lines and the corresponding primary tumor cells showed that the initial phenotype was preserved after more than 12 weeks of expansion and after cryopreservation. This proves that the generated cell lines are an accurate tool that can be used for the development of novel personalized immunotherapies. Most importantly, our results enabled the design of a translational research pipeline that allows the systematic characterization of the immune profile of understudied sarcomas for the first time in order to gain insight into which patients may directly benefit from available immunotherapies and which may be eligible to enroll in clinical trials assessing the efficacy of checkpoint inhibitors. In summary, the generated sarcoma biobank serves as a unique and highly representative primary resource that can be utilized to identify potential candidates for novel targeted and personalized immunotherapies for the treatment of sarcomas. Citation Format: Anna-Maria Georgoudaki, Robin Krueger, Michelle Hartman, Tamara Chinn, Dustin Tran, Reneé Potens, Wendy Weston, H. Thomas Temple, Adil D. Duru. Identification of prevalent targets for the development of tailored sarcoma immunotherapies using a rapid clinic-to-bench immunoprofiling pipeline [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A23.

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Sarcomas are a broad category of cancers arising from cells of mesenchymal origin that occur in bone and soft tissues. They affect humans of all ages, and standard treatment options remain ineffective at increasing overall survival. Targeted immunotherapy is a rapidly developing field that offers extremely promising therapeutic prospects for many cancers. However, sarcomas are vastly understudied in this field. Natural killer (NK) cells are immune cells with inherent tumor-killing abilities that are a promising option for cancer immunotherapy. However, tumors can dysregulate the expression of NK cells ligands to escape immune detection, and the details of these escape mechanisms are not well known in sarcomas. Thus, a better understanding of NK cell-sarcoma interactions is necessary for the development of novel immunotherapies. In order to address this, we generated 20 cell lines from primary sarcoma tumors and characterized a group of known cancer-related NK cell receptor ligands. Our results show that all primary sarcoma cell lines express the inhibitory NKp44 ligand, PCNA, as well as CD112 and/or CD155, both of which are ligands for the NK cell activating receptor DNAM-1. However, NK cell cytotoxicity against sarcoma cells is still relatively low. Therefore, we generated a cell-based screening platform in which genetically modified (GM) NK cells that overexpress one activating receptor at a time are tested for degranulation against different sarcoma cell lines (n=12). Our results indicated patient-specific involvement of multiple NK cell receptors. In line with the conserved expression of CD112 and CD155 on sarcoma cells, we observed that DNAM-1+ GM-NK cells display significantly enhanced antitumor responses against all 12 cell lines when compared to wild-type (WT)-NK cells. We further confirmed that this enhanced NK cell response was indeed DNAM-1-dependent by interfering with the CD155/DNAM-1 interaction using blocking antibodies targeting either the receptor or its ligand. Additionally, the response of DNAM-1+ versus WT-NK cells was tested against various cancer cell lines, including those derived from a metastatic prostate carcinoma (LNCaP), primary pancreatic ductal adenocarcinoma (Capan-2), primary colorectal adenocarcinoma (Caco-2), primary lung alveolar basal epithelial adenocarcinoma (A549), metastatic neuroblastoma (SH-Sy5y), metastatic nerve sheath tumor (SNF02.2), two melanomas (A375 and DM6), and two leukemias (K562 and THP-1). In conclusion, DNAM-1 overexpression elicited a dynamic increase in NK cell degranulation against all cancer cell lines tested, including those that failed to induce a notable response in WT-NK cells, supporting the broad potential applicability of the DNAM-1+ GM-NK cells for the treatment of several malignancies. Furthermore, tumors expressing CD112 and/or CD155 can be targeted by DNAM-1+ GM-NK cells, and our GM NK cell screening platform can be used for rapid screening against not only sarcomas but also several cancers. Citation Format: Ece C. Sayitoglu, Michael Chrobok, Anna-Maria Georgoudaki, Benjamin J. Josey, Michelle Hartman, Esha Vallabhaneni, Rajeev Herekar, Savannah Bergeron, Robin Krueger, Tolga Sutlu, Evren Alici, Harry T. Temple, Adil D. Duru. Natural killer cells genetically modified to overexpress DNAM-1 exert enhanced antitumor responses against CD112/CD155+ sarcomas and other malignancies [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A55.