Glyphosate, or N-phosphomethyl(glycine), is an organophosphorus compound and a competitive inhibitor of the shikimate pathway that allows aromatic amino acid biosynthesis in plants and ...microorganisms. Its utilization in broad-spectrum herbicides, such as RoundUp
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, has continued to increase since 1974; glyphosate, as well as its primary metabolite aminomethylphosphonic acid, is measured in soils, water, plants, animals and food. In humans, glyphosate is detected in blood and urine, especially in exposed workers, and is excreted within a few days. It has long been regarded as harmless in animals, but growing literature has reported health risks associated with glyphosate and glyphosate-based herbicides. In 2017, the International Agency for Research on Cancer (IARC) classified glyphosate as "probably carcinogenic" in humans. However, other national agencies did not tighten their glyphosate restrictions and even prolonged authorizations of its use. There are also discrepancies between countries' authorized levels, demonstrating an absence of a clear consensus on glyphosate to date. This review details the effects of glyphosate and glyphosate-based herbicides on fish and mammal health, focusing on the immune system. Increasing evidence shows that glyphosate and glyphosate-based herbicides exhibit cytotoxic and genotoxic effects, increase oxidative stress, disrupt the estrogen pathway, impair some cerebral functions, and allegedly correlate with some cancers. Glyphosate effects on the immune system appear to alter the complement cascade, phagocytic function, and lymphocyte responses, and increase the production of pro-inflammatory cytokines in fish. In mammals, including humans, glyphosate mainly has cytotoxic and genotoxic effects, causes inflammation, and affects lymphocyte functions and the interactions between microorganisms and the immune system. Importantly, even as many outcomes are still being debated, evidence points to a need for more studies to better decipher the risks from glyphosate and better regulation of its global utilization.
Reactive oxygen species (ROS) have an established role in inflammation and host defense, as they kill intracellular bacteria and have been shown to activate the NLRP3 inflammasome. Here, we find that ...ROS generated by mitochondrial respiration are important for normal lipopolysaccharide (LPS)-driven production of several proinflammatory cytokines and for the enhanced responsiveness to LPS seen in cells from patients with tumor necrosis factor receptor-associated periodic syndrome (TRAPS), an autoinflammatory disorder caused by missense mutations in the type 1 TNF receptor (TNFR1). We find elevated baseline ROS in both mouse embryonic fibroblasts and human immune cells harboring TRAPS-associated TNFR1 mutations. A variety of antioxidants dampen LPS-induced MAPK phosphorylation and inflammatory cytokine production. However, gp91(phox) and p22(phox) reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits are dispensable for inflammatory cytokine production, indicating that NADPH oxidases are not the source of proinflammatory ROS. TNFR1 mutant cells exhibit altered mitochondrial function with enhanced oxidative capacity and mitochondrial ROS generation, and pharmacological blockade of mitochondrial ROS efficiently reduces inflammatory cytokine production after LPS stimulation in cells from TRAPS patients and healthy controls. These findings suggest that mitochondrial ROS may be a novel therapeutic target for TRAPS and other inflammatory diseases.
Interleukin-17A (IL-17A) and IL-17F are 2 of several cytokines produced by T helper 17 cells (Th17), which are able to indirectly induce the recruitment of neutrophils. Recently, human Th17 cells ...have been phenotypically characterized and shown to express discrete chemokine receptors, including CCR2 and CCR6. Herein, we show that highly purified neutrophils cultured with interferon-γ plus lipopolysaccharide produce the CCL2 and CCL20 chemokines, the known ligands of CCR2 and CCR6, respectively. Accordingly, supernatants from activated neutrophils induced chemotaxis of Th17 cells, which was greatly suppressed by anti-CCL20 and anti-CCL2 antibodies. We also discovered that activated Th17 cells could directly chemoattract neutrophils via the release of biologically active CXCL8. Consistent with this reciprocal recruitment, neutrophils and Th17 cells were found in gut tissue from Crohn disease and synovial fluid from rheumatoid arthritis patients. Finally, we report that, although human Th17 cells can directly interact with freshly isolated or preactivated neutrophils via granulocyte-macrophage colony-stimulating factor, tumor necrosis factor-α, and interferon-γ release, these latter cells cannot be activated by IL-17A and IL-17F, because of their lack of IL-17RC expression. Collectively, our results reveal a novel chemokine-dependent reciprocal cross-talk between neutrophils and Th17 cells, which may represent a useful target for the treatment of chronic inflammatory diseases.
Innate lymphoid cells (ILC) play an important role in many immune processes, including control of infections, inflammation, and tissue repair. To date, little is known about the metabolism of ILC and ...whether these cells can metabolically adapt in response to environmental signals. Here we show that type 2 innate lymphoid cells (ILC2), important mediators of barrier immunity, predominantly depend on fatty acid (FA) metabolism during helminth infection. Further, in situations where an essential nutrient, such as vitamin A, is limited, ILC2 sustain their function and selectively maintain interleukin 13 (IL-13) production via increased acquisition and utilization of FA. Together, these results reveal that ILC2 preferentially use FAs to maintain their function in the context of helminth infection or malnutrition and propose that enhanced FA usage and FA-dependent IL-13 production by ILC2 could represent a host adaptation to maintain barrier immunity under dietary restriction.
Activation of the NLRP3 inflammasome is associated with metabolic dysfunction, and intermittent fasting has been shown to improve clinical presentation of NLRP3 inflammasome-linked diseases. As ...mitochondrial perturbations, which function as a damage-associated molecular pattern, exacerbate NLRP3 inflammasome activation, we investigated whether fasting blunts inflammasome activation via sirtuin-mediated augmentation of mitochondrial integrity.
We performed a clinical study of 19 healthy volunteers. Each subject underwent a 24-hour fast and then was fed a fixed-calorie meal. Blood was drawn during the fasted and fed states and analyzed for NRLP3 inflammasome activation. We enrolled an additional group of 8 healthy volunteers to assess the effects of the sirtuin activator, nicotinamide riboside, on NLRP3 inflammasome activation.
In the fasting/refeeding study, individuals showed less NLRP3 inflammasome activation in the fasted state compared with that in refed conditions. In a human macrophage line, depletion of the mitochondrial-enriched sirtuin deacetylase SIRT3 increased NLRP3 inflammasome activation in association with excessive mitochondrial ROS production. Furthermore, genetic and pharmacologic SIRT3 activation blunted NLRP3 activity in parallel with enhanced mitochondrial function in cultured cells and in leukocytes extracted from healthy volunteers and from refed individuals but not in those collected during fasting.
Together, our data indicate that nutrient levels regulate the NLRP3 inflammasome, in part through SIRT3-mediated mitochondrial homeostatic control. Moreover, these results suggest that deacetylase-dependent inflammasome attenuation may be amenable to targeting in human disease.
ClinicalTrials.gov NCT02122575 and NCT00442195.
Division of Intramural Research, NHLBI of the NIH.
Autosomal recessive mutations in proteasome subunit β 8 (PSMB8), which encodes the inducible proteasome subunit β5i, cause the immune-dysregulatory disease chronic atypical neutrophilic dermatosis ...with lipodystrophy and elevated temperature (CANDLE), which is classified as a proteasome-associated autoinflammatory syndrome (PRAAS). Here, we identified 8 mutations in 4 proteasome genes, PSMA3 (encodes α7), PSMB4 (encodes β7), PSMB9 (encodes β1i), and proteasome maturation protein (POMP), that have not been previously associated with disease and 1 mutation in PSMB8 that has not been previously reported. One patient was compound heterozygous for PSMB4 mutations, 6 patients from 4 families were heterozygous for a missense mutation in 1 inducible proteasome subunit and a mutation in a constitutive proteasome subunit, and 1 patient was heterozygous for a POMP mutation, thus establishing a digenic and autosomal dominant inheritance pattern of PRAAS. Function evaluation revealed that these mutations variably affect transcription, protein expression, protein folding, proteasome assembly, and, ultimately, proteasome activity. Moreover, defects in proteasome formation and function were recapitulated by siRNA-mediated knockdown of the respective subunits in primary fibroblasts from healthy individuals. Patient-isolated hematopoietic and nonhematopoietic cells exhibited a strong IFN gene-expression signature, irrespective of genotype. Additionally, chemical proteasome inhibition or progressive depletion of proteasome subunit gene transcription with siRNA induced transcription of type I IFN genes in healthy control cells. Our results provide further insight into CANDLE genetics and link global proteasome dysfunction to increased type I IFN production.
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•We optimized bioenergetic flux assays in 2D and 3D models of the mammary gland.•Estrogens and EDCs reprogram mammary epithelial cell energy metabolism.•The metabolic response and ...sensitivity to EDCs change between 2D and 3D cultures.•Organoids reproduce the morphological change observed in vivo after EDC treatment.•Past pregnancies alter the metabolic response to EDCs of mammary organoids ex vivo.
Due to its sensitivity to hormonal signaling, the mammary gland is often referred to as a sentinel organ for the study of endocrine-disrupting chemicals (EDCs), environmental pollutants that can interfere with the estrogen signaling pathway and induce mammary developmental defects. If and how EDCs impact mammary epithelial cell metabolism has not yet been documented. Herein, to study how estrogens and EDCs modulate mammary gland metabolism, we performed bioenergetic flux analyses using mouse mammary epithelial organoids compared to cells grown in monolayer culture. Several EDCs were tested, including bisphenol A (BPA), its close derivative BPS, a new BPA replacement copolyester called TritanTM, and the herbicide glyphosate. We report that estrogens reprogrammed mammary epithelial cell metabolism differently when grown in two- and three-dimensional models. Specific EDCs were also demonstrated to alter bioenergetic fluxes, thus identifying a new potential adverse effect of these molecules. Notably, organoids were more sensitive to low EDC concentrations, highlighting them as a key model for screening the impact of various environmental pollutants. Mechanistically, transcriptomic analyses revealed that EDCs interfered with the regulation of estrogen target genes and the expression of metabolic genes in organoids. Furthermore, co-treatment with the anti-estrogen fulvestrant blocked these metabolic impacts of EDCs, suggesting that, at least partially, they act through modulation of the estrogen receptor activity. Finally, we demonstrate that mammary organoids can be used for long-term studies on EDC exposure to study alterations in organogenesis/morphogenesis and that past pregnancies can modulate the sensitivity of mammary epithelial organoids to specific EDCs. Overall, this study demonstrates that estrogens and EDCs modulate mammary epithelial cell metabolism in monolayer and organoid cultures. A better understanding of the metabolic impacts of EDCs will allow a better appreciation of their adverse effects on mammary gland development and function.
Evidence accumulating over the past decade has linked alterations in bioenergetic metabolism to the pathogenesis of several diseases, including inflammatory conditions and cancer. However, the mutual ...relationship between the effector functions and the metabolism of immune cells has begun to emerge only recently. Similar to malignant cells, both innate and adaptive immune cells undergo a metabolic reprogramming that is required for effector functions, de facto underlying the elicitation of a robust immune response. These changes allow immune cells not only to rapidly respond to pathogens or (pre)malignant cells but also to adapt to changing microenvironmental conditions. Targeting the metabolic alterations of malignant cells has been the subject of an intense wave of investigation, resulting in the identification of promising therapeutic strategies. Since the inflammatory milieu and the tumor microenvironment are similar, the metabolism of immune cells and its regulation has recently come under renewed interest as a target for immunotherapy. Here, we describe different tools and techniques to study the bioenergetic metabolism of cultured cells, using immune cells as a model. Our methodological approach relies on an extracellular flux analyzer, an instrument that enables the real-time measurement of the two central pathways used by living cells to generate adenosine triphosphate: glycolysis and oxidative phosphorylation. This instrument and similar technological innovations have transformed the study of cellular metabolism, unveiling its profound impact on various immunologic and oncological disorders.
Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are stromal precursors endowed with extensive immunomodulative properties. In this study, we aimed to assess whether Toll-like receptor-3 ...(TLR3)- and TLR4-activated BM-MSC influence human neutrophil (PMN) responses under coculture conditions. We show that TLR3 triggering by polyinosinic:polycytidylic acid dramatically amplifies, in a more significant manner than TLR4 triggering by lipopolysaccharide, the antiapoptotic effects that resting BM-MSC constitutively exert on PMN under coculture conditions, preserving a significant fraction of viable and functional PMN up to 72 hours. In addition, TLR3- and TLR4-activated BM-MSC enhance respiratory burst ability and CD11b expression by PMN. The coculture in the absence of cell contact and the incubation of PMN in supernatants harvested from TLR3- and TLR4-activated BM-MSC yield comparable results in terms of increased survival and immunophenotypic changes, thus suggesting the involvement of endogenous soluble factors. Neutralizing experiments reveal that the biological effects exerted on PMN by TLR3-activated BM-MSC are mediated by the combined action of interleukin 6, interferon-β (IFN-β), and granulocyte macrophage colony-stimulating factor (GM-CSF), while those exerted by TLR4-activated BM-MSC mostly depend on GM-CSF. MSC isolated from thymus, spleen, and subcutaneous adipose tissue behaves similarly. Finally, the effects exerted by TLR3- or TLR4-stimulated BM-MSC on PMN are conserved even after the previous priming of BM-MSC with IFN-γ and tumor necrosis factor-α. Our data highlight a novel mechanism by which MSC sustain and amplify the functions of PMN in response to TLR3- and TLR4-triggering and may consequently contribute to inflammatory disorders.
Bisphenol A (BPA) and bisphenol S (BPS) are synthetic chemicals used to produce plastics which can be released in food and water. Once ingested, BPA and BPS are metabolized by the liver, mainly as ...glucuronidated metabolites, and are excreted through urine. Since urine can be stored for many hours, the bladder is chronically exposed to BP metabolites, and studies have shown that these metabolites can remain active in the organism. Therefore, the effect of physiological concentrations of glucuronidated BPs was evaluated on the bioenergetics (glycolysis and mitochondrial respiration), migration and proliferation of normal urothelial cells, and non-invasive and invasive bladder cancer cells. The results demonstrated that an exposure of 72 h to glucuronidated BPA or BPS decreased the bioenergetics and activity of normal urothelial cells, while increasing these parameters for bladder cancer cells. These findings suggest that BP metabolites are not as inactive as initially believed, and their ubiquitous presence in the urine could promote bladder cancer progression.
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