Aims The neutrophil–lymphocyte ratio (NLR) is a readily available inflammatory biomarker that may associate with atherosclerosis and predict cardiovascular (CV) events. The aims of this study are to determine whether the NLR predicts incident major adverse cardiovascular events (MACE) and is modified by anti-inflammatory therapy. Methods and results Baseline and on-treatment NLRs were calculated from complete blood counts among 60 087 participants randomized in the CANTOS, JUPITER, SPIRE-1, SPIRE-2, and CIRT trials to receive placebo or canakinumab, rosuvastatin, bococizumab, or methotrexate, respectively, and followed up for MACE. All analyses were performed first in CANTOS, and then externally validated in the other four trials. For the five trials, hazard ratios for major CV events and mortality comparing NLR quartiles were computed using Cox proportional hazards models, and the effect of each randomized intervention on the NLR was evaluated in comparison to placebo. The NLR modestly correlated with interleukin-6, C-reactive protein, and fibrinogen levels but minimally with lipids. In all five randomized trials, baseline NLR predicted incident CV events and death; the per-quartile increase in risk of MACE was 20% in CANTOS [95% confidence interval (CI) 14–25%, P < 0.0001], 31% in SPIRE-1 (95% CI 14–49%, P = 0.00007), 27% in SPIRE-2 (95% CI 12–43%, P = 0.0002), 9% in CIRT (95% CI 0.2–20%, P = 0.045), and 11% in JUPITER (95% CI 1–22%, P = 0.03). While lipid-lowering agents had no significant impact on the NLR, anti-inflammatory therapy with canakinumab lowered the NLR (P < 0.0001). Conclusion The NLR, an easily obtained inflammatory biomarker, independently predicts CV risk and all-cause mortality, and is reduced by interleukin-1β blockade with canakinumab.

Aims Targeting vascular inflammation represents a novel therapeutic approach to reduce complications of atherosclerosis. Neutralizing the pro-inflammatory cytokine interleukin-1β (IL-1β) using canakinumab, a monoclonal antibody, reduces the incidence of cardiovascular events in patients after myocardial infarction (MI). The biological basis for these beneficial effects remains incompletely understood. We sought to explore the mechanisms of IL-1β-targeted therapies. Methods and Results In mice with early atherosclerosis (ApoE-/- mice on a high-cholesterol diet for six weeks), we found that three weeks of NLRP3-inflammasome inhibition or anti-IL-1β treatment (using either MCC950, an NLRP3 inflammasome inhibitor which blocks production and release of active IL-1β; or a murine analog of canakinumab) dampened accumulation of leukocytes in atherosclerotic aortas, which consequently resulted in slower progression of atherosclerosis. Causally, we found that endothelial cells from atherosclerotic aortas lowered expression of leukocyte chemoattractants and adhesion molecules upon NLRP3-inflammasome inhibition, indicating that NLRP3-inflammasome- and IL-1β-targeted therapies reduced blood leukocyte recruitment to atherosclerotic aortas. In accord, adoptive transfer experiments revealed that anti-IL-1β treatment mitigated blood myeloid cell uptake to atherosclerotic aortas. We further report that anti-IL-1β treatment and NLRP3-inflammasome inhibition reduced inflammatory leukocyte supply by decreasing proliferation of bone marrow hematopoietic stem and progenitor cells, demonstrating that suppression of IL-1β and the NLRP3-inflammasome lowered production of disease-propagating leukocytes. Using bone marrow reconstitution experiments, we observed that hematopoietic cell-specific NLRP3-inflammasome activity contributed to both enhanced recruitment and increased supply of blood inflammatory leukocytes. Further experiments that queried whether anti-IL-1β treatment reduced vascular inflammation also in post-MI accelerated atherosclerosis documented the operation of convergent mechanisms (reduced supply and uptake of inflammatory leukocytes). In line with our pre-clinical findings, post-MI patients on canakinumab treatment showed reduced blood monocyte numbers. Conclusions Our murine and human data reveal that anti-IL-1β treatment and NLRP3-inflammasome inhibition dampened vascular inflammation and progression of atherosclerosis through reduced blood inflammatory leukocyte 1) supply and 2) uptake into atherosclerotic aortas providing additional mechanistic insights into links between hematopoiesis and atherogenesis, and into the beneficial effects of NLRP3-inflammasome- and IL-1β-targeted therapies. Translational perspective Therapeutic targeting of vascular inflammation represents a promising avenue to reduce complications of atherosclerosis. Neutralizing the pro-inflammatory cytokine interleukin-1β (IL-1β) reduces the incidence of cardiovascular events in patients with prior myocardial infarction. However, the mechanisms underlying these beneficial effects remain incompletely understood. This study explored how IL-1β and NLRP3-inflammasome suppression mitigated plaque progression. Our murine and human data reveal that pharmacological anti-IL-1β treatment and NLRP3-inflammasome inhibition dampened inflammatory leukocyte accumulation in atherosclerotic aortas through 1) decreased blood inflammatory leukocyte supply and 2) reduced blood inflammatory leukocyte uptake into in atherosclerotic aortas. These data provide additional mechanistic insights into links between hematopoiesis and atherogenesis, and inform future anti-inflammatory interventions in patients with atherosclerosis.

Preclinical models that reliably recapitulate the immunosuppressive properties of human gliomas are essential to assess immune-based therapies. GL261 murine glioma cells are widely used as a syngeneic animal model of glioma, however, it has become common practice to transfect these cells with luciferase for fluorescent tumor tracking. The aim of this study was to compare the survival of mice injected with fluorescent or non-fluorescent GL261 cells and characterize the differences in their tumor microenvironment. Mice were intracranially implanted with GL261, GL261 Red-FLuc or GL261-Luc2 cells at varying doses. Cytokine profiles were evaluated by proteome microarray and Kaplan-Meier survival analysis was used to determine survival differences. Median survival for mice implanted with 5 × 10 4 GL261 cells was 18 to 21 days. The GL261 Red-FLuc implanted mice cells did not reach median survival at any tumor dose. Mice injected with 3 × 10 5 GL261-Luc2 cells reached median survival at 23 days. However, median survival was significantly prolonged to 37 days in mice implanted with 5 × 10 4 GL261-Luc2 cells. Additionally, proteomic analyses revealed significantly elevated inflammatory cytokines in the supernatants of the GL261 Red-FLuc cells and GL261-Luc2 cells. Our data suggest that GL261 Red-FLuc and GL261-Luc2 murine models elicit an anti-tumor immune response by increasing pro-inflammatory modulators. Glioblastoma (GBM) is the most common malignant primary brain tumor in adults 1. Despite ongoing studies and numerous clinical trials, the prognosis remains poor 2. There is an urgent need to provide these patients with new therapies, as the standard of care treatment has gone unchanged for more than a decade 3. In light of the promising advances made in the area of immune therapy as a treatment for various solid cancers 4-7 , the field of neuro-oncology has embraced immunotherapies for gliomas as a promising area of preclinical and clinical investigation 8. However, recent trials like the Phase III CheckMate-143 9 and Phase III CheckMate-498 10 show poor efficacy despite promising results from pre-clinical studies 11-15. The lack of translation from preclinical to clinical studies illustrates the need for a more rigorous characterization of the pre-clinical models currently being used in the scientific community. The development of novel immune therapeutics is not possible without a reliable preclinical animal model that accurately mimics the complex immune landscape of GBM. Of the various classes of preclinical mouse models, syngeneic models have been indispensable for evaluating immune therapies in GBM 16. Syngeneic murine models are models that rely on allografts of immortalized cancer cells from the same mouse strain from which the model originates.