The receptor for advanced glycation end products promotes pancreatic carcinogenesis and accumulation of myeloid-derived suppressor cells.

Pancreatic ductal adenocarcinoma (PDA) has an aggressive natural history and is resistant to therapy. The receptor for advanced glycation end products (RAGE) is a pattern recognition receptor for many damage-associated molecular pattern molecules. RAGE is overexpressed in both human and murine models of PDA as well as most advanced epithelial neoplasms. The immunosuppressive nature of the PDA microenvironment is facilitated, in part, by the accumulation of regulatory immune cell infiltrates such as myeloid-derived suppressor cells (MDSCs). To study the role of RAGE expression in the setting of mutant Ras-promoted pancreatic carcinogenesis (KC), a triple-transgenic model of spontaneous murine PDA in a RAGE-null background (KCR) was generated. KCR mice had markedly delayed pancreatic carcinogenesis and a significant diminution of MDSCs compared with KC mice at comparable time points postweaning. Although RAGE was not required for the development or suppressor activity of MDSCs, its absence was associated with temporally limited pancreatic neoplasia and altered phenotype and function of the myeloid cells. In lieu of MDSCs, KCR animals at comparable time points exhibited mature CD11b(+)Gr1(-)F4/80(+) cells that were not immunosuppressive in vitro. KCR mice also maintained a significantly less suppressive milieu evidenced by marked decreases in CCL22 in relation to CXCL10 and diminished serum levels of IL-6.

Nonhuman Primate (Rhesus Macaque) Models of Severe Pressure-Targeted Hemorrhagic and Polytraumatic Hemorrhagic Shock.

BACKGROUND: We endeavored to develop clinically translatable nonhuman primate (NHP) models of severe polytraumatic hemorrhagic shock. METHODS: NHPs were randomized into five severe pressure-targeted hemorrhagic shock (PTHS) ±â€Šadditional injuries scenarios: 30-min PTHS (PTHS-30), 60-min PTHS (PTHS-60), PTHS-60 + soft tissue injury (PTHS-60+ST), PTHS-60+ST + femur fracture (PTHS-60+ST+FF), and decompensated PTHS+ST+FF (PTHS-D). Physiologic parameters were recorded and blood samples collected at five time points with animal observation through T = 24 h. Results presented as mean ±â€ŠSEM; statistics: log transformation followed by two-way ANOVA with Bonferroni multiple comparisons, Wilcoxon nonparametric test for comparisons, and the Friedmans' one-way ANOVA; significance: P < 0.05. RESULTS: Percent blood loss was 40% ±â€Š2, 59% ±â€Š3, 52% ±â€Š3, 49% ±â€Š2, and 54% ±â€Š2 for PTHS-30, PTHS-60, PTHS-60+ST, PTHS-60+ST+FF, and PTHS-D, respectively. All animals survived to T = 24 h except one in each of the PTHS-60 and PTHS-60+ST+FF groups and seven in the PTHS-D group. Physiologic, coagulation, and inflammatory parameters demonstrated increasing derangements with increasing model severity. CONCLUSION: NHPs exhibit a high degree of resilience to hemorrhagic shock and polytrauma as evidenced by moderate perturbations in metabolic, coagulation, and immunologic outcomes with up to 60 min of profound hypotension regardless of injury pattern. Extending the duration of PTHS to the point of decompensation in combination with polytraumatic injury, evoked derangements consistent with those observed in severely injured trauma patients which would require ICU care. Thus, we have successfully established a clinically translatable NHP trauma model for use in testing therapeutic interventions to trauma.

Severe Hemorrhagic Shock Induces Acute Activation and Expansion of IL-8+/IL-10+ Neutrophils with Enhanced Oxidative Reactivity in Non-Human Primates.

BACKGROUND: Neutrophilic inflammation is a mediator of morbidity and mortality in response to hemorrhagic shock. Although injury-induced neutrophil margination has long been observed, the nature of neutrophils' role in the "second hit" paradigm remains to be fully elucidated. We sought to extensively characterize neutrophil phenotype and functionality in response to severe hemorrhage in non-human primates (NHPs). METHODS: NHPs (n = 8) were subjected to severe hemorrhagic shock and resuscitation. Blood was obtained at baseline (T = 0 min), end of shock (T = 60 min), end of resuscitation (T = 180 min), T = 360 min, and 24 h (T = 1440 min). Neutrophils were quantified by complete blood count and flow cytometry. IL-8 and IL-10 production was determined by intracellular flow cytometry. Oxidation of dihydrorhodamine-123 (DHR-123) was used to determine neutrophil oxidative bursts (untreated), priming (+fMLP), and burst capacity (+PMA/ionomycin) via microplate reader ex vivo. Data are reported as mean ±â€ŠSEM; statistical significance was measured using repeated measures ANOVA with Bonferroni adjustment. P < 0.05 is considered significant. RESULTS: CD45CD11bCD16 neutrophils doubled postinjury (P < 0.0001); this was due to activated IL-8/IL-10 neutrophils that increased in frequency in relation to resting IL-8IL-10 cells. At 24 h, the proportions of activated to resting neutrophils returned to baseline levels. Resuscitative measures initially decreased neutrophil oxidative output; however, oxidative bursts, priming, and burst capacity were significantly increased at 24 h (P < 0.0025, 0.0124, and 0.0118, respectively). CONCLUSION: These results demonstrate an acute expansion and phenotypic activation of circulating neutrophils postinjury followed by a return to homeostatic proportions within 24 h; paradoxically, phenotypically "resting" neutrophils at 24 h have significantly higher oxidative potential, predisposing for exaggerated inflammatory responses. These data are consistent with clinical literature and provide important functional insight into neutrophil-mediated shock pathology.

Rapid Detection of Neutrophil Oxidative Burst Capacity is Predictive of Whole Blood Cytokine Responses.

BACKGROUND: Maladaptive immune responses, particularly cytokine and chemokine-driven, are a significant contributor to the deleterious inflammation present in many types of injury and infection. Widely available applications to rapidly assess individual inflammatory capacity could permit identification of patients at risk for exacerbated immune responses and guide therapy. Here we evaluate neutrophil oxidative burst (NOX) capacity measured by plate reader to immuno-type Rhesus Macaques as an acute strategy to rapidly detect inflammatory capacity and predict maladaptive immune responses as assayed by cytokine array. METHODS: Whole blood was collected from anesthetized Rhesus Macaques (n = 25) and analyzed for plasma cytokine secretion (23-plex Luminex assay) and NOX capacity. For cytokine secretion, paired samples were either unstimulated or ex-vivo lipopolysaccharide (LPS)-stimulated (100µg/mL/24h). NOX capacity was measured in dihydrorhodamine-123 loaded samples following phorbol 12-myristate 13-acetate (PMA)/ionomycin treatment. Pearson's test was utilized to correlate NOX capacity with cytokine secretion, p<0.05 considered significant. RESULTS: LPS stimulation induced secretion of the inflammatory molecules G-CSF, IL-1ß, IL-1RA, IL-6, IL-10, IL-12/23(p40), IL-18, MIP-1α, MIP-1ß, and TNFα. Although values were variable, several cytokines correlated with NOX capacity, p-values≤0.0001. Specifically, IL-1ß (r = 0.66), IL-6 (r = 0.74), the Th1-polarizing cytokine IL-12/23(p40) (r = 0.78), and TNFα (r = 0.76) were strongly associated with NOX. CONCLUSION: NOX capacity correlated with Th1-polarizing cytokine secretion, indicating its ability to rapidly predict inflammatory responses. These data suggest that NOX capacity may quickly identify patients at risk for maladaptive immune responses and who may benefit from immuno-modulatory therapies. Future studies will assess the in-vivo predictive value of NOX in animal models of immune-mediated pathologies.

Eat-me: autophagy, phagocytosis, and reactive oxygen species signaling.

SIGNIFICANCE: Phagocytosis is required for the clearance of dying cells. The subsequent regulation of inflammatory responses by phagocytic cells is mediated by both innate and adaptive immune responses. Autophagy, an evolutionarily ancient process of lysosomal self-digestion of organelles, protein aggregates, apoptotic corpses, and cytosolic pathogens, has only recently become appreciated for its dynamic relationship with phagocytosis, including newly discovered autophagic-phagocytosis "hybrid" processes such as microtubule-associated protein 1 light chain 3-associated phagocytosis (LAP). RECENT ADVANCES: Signal transduction by reactive oxygen species (ROS) plays a critical role in the modulation of autophagy, phagocytosis, and LAP, and serves as both a link and an additional layer of regulation between these processes. Furthermore, specific targets for oxidation by ROS molecules have recently begun to become identified in each of these processes, as have "shared" proteins that facilitate the successful completion of both autophagy and phagocytosis. High mobility group box 1 is at the crossroads of autophagy, phagocytosis, and oxidative stress. CRITICAL ISSUES: In this review, we discuss the most recent findings that link elements of autophagy and phagocytosis, specifically through redox-dependent signal transduction. These interconnected cellular processes are placed in the context of cell death and immunity in both health and disease. FUTURE DIRECTIONS: Given the broad roles that autophagy, phagocytosis, and ROS signaling play in human health, disease, and the maintenance of cellular and organismal homeostatic balance, it is important to delineate intersections between these pathways and uncover targets for potential therapeutic intervention in the setting of autoimmune and inflammatory diseases.

The myeloid response to pancreatic carcinogenesis is regulated by the receptor for advanced glycation end-products.

We identified a critical role for receptor for advanced glycation end products (RAGE) in the intratumoral accumulation of myeloid-derived suppressor cells (MDSCs) during pancreatic carcinogenesis. The absence of RAGE markedly delayed neoplasia and limited MDSC accumulation in mice expressing an oncogenic variant of Kras. In spite of MDSCs, these mice accumulated non-immunosuppressive macrophages. Thus, RAGE regulates carcinogenesis and consequent myeloid responses.