Diesel Exhaust Particle (DEP)-induced glucose intolerance is driven by an intestinal innate immune response and NLRP3 activation in mice.

BACKGROUND: We previously found that air pollution particles reaching the gastrointestinal tract elicit gut inflammation as shown by up-regulated gene expression of pro-inflammatory cytokines and monocyte/macrophage markers. This inflammatory response was associated with beta-cell dysfunction and glucose intolerance. So far, it remains unclear whether gut inflammatory changes upon oral air pollution exposure are causally linked to the development of diabetes. Hence, our aim was to assess the role of immune cells in mediating glucose intolerance instigated by orally administered air pollutants. METHODS: To assess immune-mediated mechanisms underlying air pollution-induced glucose intolerance, we administered diesel exhaust particles (DEP; NIST 1650b, 12 µg five days/week) or phosphate-buffered saline (PBS) via gavage for up to 10 months to wild-type mice and mice with genetic or pharmacological depletion of innate or adaptive immune cells. We performed unbiased RNA-sequencing of intestinal macrophages to elucidate signaling pathways that could be pharmacologically targeted and applied an in vitro approach to confirm these pathways. RESULTS: Oral exposure to air pollution particles induced an interferon and inflammatory signature in colon macrophages together with a decrease of CCR2- anti-inflammatory/resident macrophages. Depletion of macrophages, NLRP3 or IL-1ß protected mice from air pollution-induced glucose intolerance. On the contrary, Rag2-/- mice lacking adaptive immune cells developed pronounced gut inflammation and glucose intolerance upon oral DEP exposure. CONCLUSION: In mice, oral exposure to air pollution particles triggers an immune-mediated response in intestinal macrophages that contributes to the development of a diabetes-like phenotype. These findings point towards new pharmacologic targets in diabetes instigated by air pollution particles.

Lung versus gut exposure to air pollution particles differentially affect metabolic health in mice.

BACKGROUND: Air pollution has emerged as an unexpected risk factor for diabetes. However, the mechanism behind remains ill-defined. So far, the lung has been considered as the main target organ of air pollution. In contrast, the gut has received little scientific attention. Since air pollution particles can reach the gut after mucociliary clearance from the lungs and through contaminated food, our aim was to assess whether exposure deposition of air pollution particles in the lung or the gut drive metabolic dysfunction in mice. METHODS: To study the effects of gut versus lung exposure, we exposed mice on standard diet to diesel exhaust particles (DEP; NIST 1650b), particulate matter (PM; NIST 1649b) or phosphate-buffered saline by either intratracheal instillation (30 µg 2 days/week) or gavage (12 µg 5 days/week) over at least 3 months (total dose of 60 µg/week for both administration routes, equivalent to a daily inhalation exposure in humans of 160 µg/m3 PM2.5) and monitored metabolic parameters and tissue changes. Additionally, we tested the impact of the exposure route in a "prestressed" condition (high-fat diet (HFD) and streptozotocin (STZ)). RESULTS: Mice on standard diet exposed to particulate air pollutants by intratracheal instillation developed lung inflammation. While both lung and gut exposure resulted in increased liver lipids, glucose intolerance and impaired insulin secretion was only observed in mice exposed to particles by gavage. Gavage with DEP created an inflammatory milieu in the gut as shown by up-regulated gene expression of pro-inflammatory cytokines and monocyte/macrophage markers. In contrast, liver and adipose inflammation markers were not increased. Beta-cell secretory capacity was impaired on a functional level, most likely induced by the inflammatory milieu in the gut, and not due to beta-cell loss. The differential metabolic effects of lung and gut exposures were confirmed in a "prestressed" HFD/STZ model. CONCLUSIONS: We conclude that separate lung and gut exposures to air pollution particles lead to distinct metabolic outcomes in mice. Both exposure routes elevate liver lipids, while gut exposure to particulate air pollutants specifically impairs beta-cell secretory capacity, potentially instigated by an inflammatory milieu in the gut.

Optimized full-spectrum flow cytometry panel for deep immunophenotyping of murine lungs.

The lung immune system consists of both resident and circulating immune cells that communicate intricately. The immune system is activated by exposure to bacteria and viruses, when cancer initiates in the lung (primary lung cancer), or when metastases of other cancer types, including breast cancer, spread to and develop in the lung (secondary lung cancer). Thus, in these pathological situations, a comprehensive and quantitative assessment of changes in the lung immune system is of paramount importance for understanding mechanisms of infectious diseases, lung cancer, and metastasis but also for developing efficacious treatments. Unfortunately, lung tissue exhibits high autofluorescence, and this high background signal makes high-parameter flow cytometry analysis complicated. Here, we provide an optimized 30-parameter antibody panel for the analysis of all major immune cell types and states in normal and metastatic murine lungs using spectral flow cytometry.

Protocol for isolation and spectral flow cytometry analysis of immune cells from the murine exocrine and endocrine pancreas.

Diabetes mellitus is a disease of the hormone-secreting endocrine pancreas. However, increasing evidence suggests that the exocrine pancreas is also involved in the pathogenesis of diabetes. In this protocol, we describe how to harvest both isolated islets and exocrine tissue from one mouse pancreas, followed by a detailed explanation of how to isolate and analyze immune cells using full-spectrum flow cytometry.

N-acetylcysteine overcomes NF1 loss-driven resistance to PI3Kα inhibition in breast cancer.

A genome-wide PiggyBac transposon-mediated screen and a resistance screen in a PIK3CAH1047R-mutated murine tumor model reveal NF1 loss in mammary tumors resistant to the phosphatidylinositol 3-kinase α (PI3Kα)-selective inhibitor alpelisib. Depletion of NF1 in PIK3CAH1047R breast cancer cell lines and a patient-derived organoid model shows that NF1 loss reduces sensitivity to PI3Kα inhibition and correlates with enhanced glycolysis and lower levels of reactive oxygen species (ROS). Unexpectedly, the antioxidant N-acetylcysteine (NAC) sensitizes NF1 knockout cells to PI3Kα inhibition and reverts their glycolytic phenotype. Global phospho-proteomics indicates that combination with NAC enhances the inhibitory effect of alpelisib on mTOR signaling. In public datasets of human breast cancer, we find that NF1 is frequently mutated and that such mutations are enriched in metastases, an indication for which use of PI3Kα inhibitors has been approved. Our results raise the attractive possibility of combining PI3Kα inhibition with NAC supplementation, especially in patients with drug-resistant metastases associated with NF1 loss.

Feed-forward loops between metastatic cancer cells and their microenvironment-the stage of escalation.

Breast cancer is the most frequent cancer among women, and metastases in distant organs are the leading cause of the cancer-related deaths. While survival of early-stage breast cancer patients has increased dramatically, the 5-year survival rate of metastatic patients has barely improved in the last 20 years. Metastases can arise up to decades after primary tumor resection, hinting at microenvironmental factors influencing the sudden outgrowth of disseminated tumor cells (DTCs). This review summarizes how the environment of the most common metastatic sites (lung, liver, bone, brain) is influenced by the primary tumor and by the varying dormancy of DTCs, with a special focus on how established metastases persist and grow in distant organs due to feed-forward loops (FFLs). We discuss in detail the importance of FFL of cancer cells with their microenvironment including the secretome, interaction with specialized tissue-specific cells, nutrients/metabolites, and that novel therapies should target not only the cancer cells but also the tumor microenvironment, which are thick as thieves.

Targeting colonic macrophages improves glycemic control in high-fat diet-induced obesity.

The obesity epidemic continues to worsen worldwide. However, the mechanisms initiating glucose dysregulation in obesity remain poorly understood. We assessed the role that colonic macrophage subpopulations play in glucose homeostasis in mice fed a high-fat diet (HFD). Concurrent with glucose intolerance, pro-inflammatory/monocyte-derived colonic macrophages increased in mice fed a HFD. A link between macrophage numbers and glycemia was established by pharmacological dose-dependent ablation of macrophages. In particular, colon-specific macrophage depletion by intrarectal clodronate liposomes improved glucose tolerance, insulin sensitivity, and insulin secretion capacity. Colonic macrophage activation upon HFD was characterized by an interferon response and a change in mitochondrial metabolism, which converged in mTOR as a common regulator. Colon-specific mTOR inhibition reduced pro-inflammatory macrophages and ameliorated insulin secretion capacity, similar to colon-specific macrophage depletion, but did not affect insulin sensitivity. Thus, pharmacological targeting of colonic macrophages could become a potential therapy in obesity to improve glycemic control.

Obesity in Humans Is Characterized by Gut Inflammation as Shown by Pro-Inflammatory Intestinal Macrophage Accumulation.

Chronic low-grade inflammation is a hallmark of obesity and associated with cardiovascular complications. However, it remains unclear where this inflammation starts. As the gut is constantly exposed to food, gut microbiota, and metabolites, we hypothesized that mucosal immunity triggers an innate inflammatory response in obesity. We characterized five distinct macrophage subpopulations (P1-P5) along the gastrointestinal tract and blood monocyte subpopulations (classical, non-classical, intermediate), which replenish intestinal macrophages, in non-obese (BMI<27kg/m2) and obese individuals (BMI>32kg/m2). To elucidate factors that potentially trigger gut inflammation, we correlated these subpopulations with cardiovascular risk factors and lifestyle behaviors. In obese individuals, we found higher pro-inflammatory macrophages in the stomach, duodenum, and colon. Intermediate blood monocytes were also increased in obesity, suggesting enhanced recruitment to the gut. We identified unhealthy lifestyle habits as potential triggers of gut and systemic inflammation (i.e., low vegetable intake, high processed meat consumption, sedentary lifestyle). Cardiovascular risk factors other than body weight did not affect the innate immune response. Thus, obesity in humans is characterized by gut inflammation as shown by accumulation of pro-inflammatory intestinal macrophages, potentially via recruited blood monocytes. Understanding gut innate immunity in human obesity might open up new targets for immune-modulatory treatments in metabolic disease.