Enhanced Proinflammatory Cytokine Production and Immunometabolic Impairment of NK Cells Exposed to Mycobacterium tuberculosis and Cigarette Smoke

AimSmoker COPD patients with chest radiological signs of prior tuberculosis (TB) showed more severe lung damage, but the mechanisms remain unclear. Emerging evidence has implicated NK cells in the pathogenesis of both COPD and TB. The purpose of this study was to delineate the profile and cytokine production of NK-cell subpopulations and their immunometabolic changes after exposure to both cigarette smoke (CS) and Mycobacterium tuberculosis(MTB).MethodsWe profiled NK-cell subpopulations in terms of percentage and cytokine production by flow cytometry in smoker patients with pulmonary TB (PTB). In an in vitro coexposure model, we investigated proinflammatory cytokine production, glycolytic influx, and oxidative phosphorylation of NK cells under CS extract (CSE) and PPD costimulation.ResultsPeripheral blood NK cells in smoker patients with active PTB (CS+PTB group) showed altered proportion of subpopulations and excessive proinflammatory cytokine expressions. In vitro, CSE- and PPD-coexposed NK-92 cells displayed enhanced proinflammatory cytokine production, concurrent with decreased glycolytic influx and oxidative phosphorylation.ConclusionSmoker patients with active PTB showed enhanced proinflammatory cytokine expression within altered NK cell subpopulations. CSE and PPD coexposure induced heightened cytokine production concurrent with impaired cell metabolism in NK cells. These novel data suggest a potential role of NK cells in the pathogenesis of lung injury in subjects with coexposure to CS and TB.

Homophilic Interaction of CD147 Promotes IL-6-Mediated Cholangiocarcinoma Invasion via the NF-κB-Dependent Pathway

Cholangiocarcinoma (CCA), an aggressive cancer of bile ducts, is a well-known chronic inflammation-related disease. The major impediment in CCA treatment is limited treatment options for advanced disease; hence, an alternative is urgently required. The role of CD147 on cytokine production has been observed in inflammation-related diseases, but not in CCA. Therefore, this study was focused on CD147-promoting proinflammatory cytokine production and functions. Proinflammatory cytokine profiles were compared between CD147 expressing CCA cells and CD147 knockout cells (CD147 KO). Three cytokines, namely interleukin (IL)-6, IL-8, and granulocyte–monocyte colony-stimulating factor (GM-CSF), were dramatically diminished in CD147 KO clones. The involvement of the CD147-related cytokines in CCA invasion was established. CD147-promoted IL-6, IL-8, and GM-CSF secretions were regulated by NF-κB nuclear translocation, Akt activation, and p38 phosphorylation. CD147-fostering IL-6 production was dependent on soluble CD147, CD147 homophilic interaction, and NF-κB function. The overexpression of specific genes in CCA tissues compared to normal counterparts emphasized the clinical importance of these molecules. Altogether, CD147-potentiated proinflammatory cytokine production leading to CCA cell invasion is shown for the first time in the current study. This suggests that modulation of CD147-related inflammation might be a promising choice for advanced CCA treatment.

MED1 Deficiency in Macrophages Accelerates Intimal Hyperplasia via ROS Generation and Inflammation

Mediator complex subunit 1 (MED1) is a component of the mediator complex and functions as a coactivator involved in the regulated transcription of nearly all RNA polymerase II-dependent genes. Previously, we showed that MED1 in macrophages has a protective effect on atherosclerosis; however, the effect of MED1 on intimal hyperplasia and mechanisms regulating proinflammatory cytokine production after macrophage MED1 deletion are still unknown. In this study, we report that MED1 macrophage-specific knockout (MED1ΔMac) mice showed aggravated neointimal hyperplasia, vascular smooth muscle cells (VSMCs), and macrophage accumulation in injured arteries. Moreover, MED1ΔMac mice showed increased proinflammatory cytokine production after an injury to the artery. After lipopolysaccharide (LPS) treatment, MED1ΔMac macrophages showed increased generation of reactive oxygen species (ROS) and reduced expression of peroxisome proliferative activated receptor gamma coactivator-1α (PGC1α) and antioxidant enzymes, including catalase and glutathione reductase. The overexpression of PGC1α attenuated the effects of MED1 deficiency in macrophages. In vitro, conditioned media from MED1ΔMac macrophages induced more proliferation and migration of VSMCs. To explore the potential mechanisms by which MED1 affects inflammation, macrophages were treated with BAY11-7082 before LPS treatment, and the results showed that MED1ΔMac macrophages exhibited increased expression of phosphorylated-p65 and phosphorylated signal transducer and activator of transcription 1 (p-STAT1) compared with the control macrophages, suggesting the enhanced activation of NF-κB and STAT1. In summary, these data showed that MED1 deficiency enhanced inflammation and the proliferation and migration of VSMCs in injured vascular tissue, which may result from the activation of NF-κB and STAT1 due to the accumulation of ROS.

862 Dectin-2, a novel target for tumor macrophage reprogramming in cancer immunotherapy

BackgroundTumor-associated macrophages (TAMs) are an abundant immune cell population in most cancers that support tumor progression through their immunosuppressive effects. We discovered that TAMs express the pattern recognition receptor Dectin-2 (Clec4n/CLEC6A), an activating C-type lectin receptor (CLR) that binds to high-mannose glycans on fungi and other microbes and induces protective immune responses against infectious disease. Dectin-2 is selectively expressed by myeloid cells, and upon ligation mediates enhanced phagocytosis, antigen processing and presentation, and proinflammatory cytokine production. Given these properties, we evaluated the therapeutic potential of targeting Dectin-2 using naturally derived ligands. We also generated human Dectin-2-targeted agonistic antibodies capable of robustly activating immunosuppressive ”M2” or TAM-like macrophages.MethodsDectin-2 expression was assessed by flow cytometry, immunohistochemistry, and using public databases. Mouse and human monocytes were differentiated into macrophages using recombinant cytokines or tumor-conditioned media, and stimulation was measured following overnight incubation with Dectin-2 ligands or antibodies. Mouse tumor cell lines were implanted into syngeneic hosts and mice were treated with mannan derived from S. cerevisiae via IT or IV administration.ResultsDectin-2 gene expression is minimal in normal human tissues but elevated across many tumor types, including breast, colon, lung, and kidney cancers. Dectin-2 is strongly expressed by macrophages differentiated in vitro and on primary TAMs. The fungal Dectin-2 ligand mannan stimulated proinflammatory cytokine production (e.g. TNFalpha) and costimulatory molecule expression (e.g. CD86) by macrophages in a Dectin-2-dependent manner. Treatment of tumor-bearing mice with mannan mediated tumor regression in multiple syngeneic tumor models, with high rates of tumor clearance in the MB49 bladder cancer model. These effects were Dectin-2 dependent, as efficacy was not observed when a Dectin-2-blocking antibody was co-administered or in knockout mice lacking Dectin-2 signaling components. Furthermore, depletion of either macrophages or T cells impaired efficacy, suggesting that Dectin-2-stimulated TAMs augment anti-tumor T cell responses. Based on these data, we developed novel Dectin-2 targeted agonist antibodies capable of activating human ”M2” or TAM-like macrophages in vitro to produce an array of proinflammatory cytokines and chemokines akin to tumor-destructive ”M1” macrophages.ConclusionsThe data presented demonstrate the therapeutic potential of targeting Dectin-2 using natural ligands or agonistic antibodies as a novel pan-cancer approach for myeloid cell-directed tumor immunotherapy.Ethics ApprovalAll animal studies were performed in accordance with Institutional Animal Care and Use Committee (IACUC)-approved protocols.

Aeromonas sobria Induces Proinflammatory Cytokines Production in Mouse Macrophages via Activating NLRP3 Inflammasome Signaling Pathways

Aeromonas sobria, a common conditional pathogenic bacteria, is widely distributed in the environment and causes gastroenteritis in humans or septicemia in fish. Of all Aeromonas species, A. sobria is the most frequently isolated from human infections especially in immunocompromised subjects. Innate immunity is the first protection system of organism to resist non-specific pathogens invasion; however, the immune response process of hosts against A. sobria infection re\mains unexplored. The present study established an A. sobria infection model using primary mouse peritoneal macrophages (PMφs). The adherence and cytotoxicity of A. sobria on PMφs were determined by May-Grünwald Giemsa staining and LDH release measurement. Pro-inflammatory cytokine expression levels were measured using qPCR, western blotting, and ELISA methods. We also investigated the levels of ASC oligomerization and determined the roles of active caspase-1 in IL-1β secretion through inhibition assays and explored the activated pattern recognition receptors through immunofluorescence. We further elucidated the roles of activated inflammasome in regulating the host’s inflammatory response through inhibition combined with ELISA assays. Our results showed that A. sobria induced lytic cell death and LDH release, whereas it had no adhesive properties on PMφs. A. sobria triggered various proinflammatory cytokine transcription level upregulation, and IL-1β occupied the highest levels. The pro-IL-1β protein expression levels increased in a dose-dependent manner with MOI ranging from 1 to 100. This process was regulated by ASC-dependent inflammasome, which cleavage pro-IL-1β into active IL-1β p17 with activated caspase-1 p20. Meanwhile, the expression levels of NLRP3 receptor significantly increased, location analysis revealed puncta-like surrounding nuclear, and inhibition of NLRP3 inflammasome downregulated caspase-1 activation and IL-1β secretion. Blocking of NLRP3 inflammasome activation through K+ efflux and cathepsin B or caspase approaches downregulated A. sobria–induced proinflammatory cytokine production. Overall, these data indicated that A. sobria induced proinflammatory cytokine production in PMφs through activating NLRP3 inflammasome signaling pathways.