The intestinal microflora, typically equated with bacteria, influences diseases such as obesity and inflammatory bowel disease. Here, we show that the mammalian gut contains a rich fungal community ...that interacts with the immune system through the innate immune receptor Dectin-1. Mice lacking Dectin-1 exhibited increased susceptibility to chemically induced colitis, which was the result of altered responses to indigenous fungi. In humans, we identified a polymorphism in the gene for Dectin-1 (CLEC7A) that is strongly linked to a severe form of ulcerative colitis. Together, our findings reveal a eukaryotic fungal community in the gut (the "mycobiome") that coexists with bacteria and substantially expands the repertoire of organisms interacting with the intestinal immune system to influence health and disease.
Innate immune cells must be able to distinguish between direct binding to microbes and detection of components shed from the surface of microbes located at a distance. Dectin-1 (also known as CLEC7A) ...is a pattern-recognition receptor expressed by myeloid phagocytes (macrophages, dendritic cells and neutrophils) that detects β-glucans in fungal cell walls and triggers direct cellular antimicrobial activity, including phagocytosis and production of reactive oxygen species (ROS). In contrast to inflammatory responses stimulated upon detection of soluble ligands by other pattern-recognition receptors, such as Toll-like receptors (TLRs), these responses are only useful when a cell comes into direct contact with a microbe and must not be spuriously activated by soluble stimuli. In this study we show that, despite its ability to bind both soluble and particulate β-glucan polymers, Dectin-1 signalling is only activated by particulate β-glucans, which cluster the receptor in synapse-like structures from which regulatory tyrosine phosphatases CD45 and CD148 (also known as PTPRC and PTPRJ, respectively) are excluded (Supplementary Fig. 1). The 'phagocytic synapse' now provides a model mechanism by which innate immune receptors can distinguish direct microbial contact from detection of microbes at a distance, thereby initiating direct cellular antimicrobial responses only when they are required.
Degradation of Gram-positive bacterial cell wall peptidoglycan in macrophage and dendritic cell phagosomes leads to activation of the NLRP3 inflammasome, a cytosolic complex that regulates processing ...and secretion of interleukin (IL)-1β and IL-18. While many inflammatory responses to peptidoglycan are mediated by detection of its muramyl dipeptide component in the cytosol by NOD2, we report here that NLRP3 inflammasome activation is caused by release of N-acetylglucosamine that is detected in the cytosol by the glycolytic enzyme hexokinase. Inhibition of hexokinase by N-acetylglucosamine causes its dissociation from mitochondria outer membranes, and we found that this is sufficient to activate the NLRP3 inflammasome. In addition, we observed that glycolytic inhibitors and metabolic conditions affecting hexokinase function and localization induce inflammasome activation. While previous studies have demonstrated that signaling by pattern recognition receptors can regulate metabolic processes, this study shows that a metabolic enzyme can act as a pattern recognition receptor.
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•Peptidoglycan-derived N-acetylglucosamine activates the NLRP3 inflammasome•N-acetylglucosamine in the cytosol is detected by hexokinase•Hexokinase release from mitochondrial outer membranes triggers NLRP3 activation•Metabolic conditions affecting hexokinase activity trigger inflammasome formation
The metabolic enzyme hexokinase unexpectedly acts as a pattern recognition receptor that recognizes bacterial peptidoglycan and triggers activation of inflammasomes.
Humans do not usually develop effective immunity to Staphylococcus aureus reinfection. Using a murine model that mimics human infection, we show that lack of protective immunity to S. aureus systemic ...reinfection is associated with robust interleukin-10 (IL-10) production and impaired protective Th17 responses. In dendritic cell co-culture assays, priming with S. aureus promotes robust T cell proliferation, but limits Th cells polarization and production of IL-1β and other cytokines important for Th1 and Th17 differentiation. We show that O-acetylation of peptidoglycan, a mechanism utilized by S. aureus to block bacterial cell wall breakdown, limits the induction of pro-inflammatory signals required for optimal Th17 polarization. IL-10 deficiency in mice restores protective immunity to S. aureus infection, and adjuvancy with a staphylococcal peptidoglycan O-acetyltransferase mutant reduces IL-10, increases IL-1β, and promotes development of IL-17-dependent, Th cell-transferable protective immunity. Overall, our study suggests a mechanism whereby S. aureus modulates cytokines critical for induction of protective Th17 immunity.
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•Protective T cell memory is not induced after systemic S. aureus infections•S. aureus induces proliferation but limits polarization of Th1 and Th17 cells•O-Acetylation of peptidoglycan represses cytokines required for Th17 polarization•Adjuvancy with ΔoatA S. aureus promotes protective memory to reinfection
Humans do not develop robust protective immunity to S. aureus infection. Sanchez et al. show that mice, like humans, do not develop a protective Th17 response after bloodstream S. aureus infection. S. aureus, via modification of cell wall peptidoglycan, limits priming of Th17 cells and blocks development of protective immunity.
Signaling by innate immune receptors initiates and orchestrates the overall immune responses to infection. Macrophage receptors recognizing pathogens can be broadly grouped into surface receptors and ...receptors restricted to intracellular compartments, such as phagosomes and the cytoplasm. There is an expectation that ingestion and degradation of microorganisms by phagocytes contributes to activation of intracellular innate receptors, although direct demonstrations of this are rare, and many model ligands are studied in soluble form, outside of their microbial context. By comparing a wild-type strain of Staphylococcus aureus and a lysozyme-sensitive mutant, we have been able directly to address the role of degradation of live bacteria by mouse macrophages in determining the overall innate cellular inflammatory response. Our investigations revealed a biphasic response to S. aureus that consisted of an initial signal resulting from the engagement of surface TLR2, followed by a later, second wave on inflammatory gene induction. This second wave of inflammatory signaling was dependent on and correlated with the timing of bacterial degradation in phagosomes. We found that TLR2 signaling followed by TLR2/TLR9 signaling enhanced sensitivity to small numbers of bacteria. We further found that treating wild-type bacteria with the peptidoglycan synthesis-inhibiting antibiotic vancomycin made S. aureus more susceptible to degradation and resulted in increased inflammatory responses, similar to those observed for mutant degradation-sensitive bacteria.
Ethanol metabolism by pancreatic acinar cells and the role of its metabolites in ethanol toxicity to the pancreas remain largely unknown. Here, we characterize ethanol metabolism in pancreatic acinar ...cells and determine the effects of ethanol metabolites on nuclear factor κB (NF-κB) and activator protein (AP)-1, transcription factors that are activated in pancreatitis and mediate expression of inflammatory molecules critical for this disease.
We measured activities of fatty acid ethyl ester (FAEE) synthase and alcohol dehydrogenase (ADH), as well as accumulation of ethanol metabolites. We measured the effects of ethanol and its metabolites on NF-κB and AP-1 activation by using a gel shift assay. Results: Pancreas metabolizes ethanol via both oxidative and nonoxidative pathways. Acinar cells are the main source of ethanol metabolism in the pancreas. Compared with the liver, FAEE synthase activity in the pancreas is greater, whereas that of ADH is much less. FAEEs activated NF-κB and AP-1, whereas acetaldehyde inhibited NF-κB activation. Ethanol decreased NF-κB binding activity in acinar cells, which was potentiated by cyanamide.
Oxidative and nonoxidative ethanol metabolites regulate transcription factors differently in pancreatic acinar cells. Ethanol may regulate NF-κB and AP-1 positively or negatively, depending on which metabolic pathway's effect predominates. These regulatory mechanisms may play a role in ethanol toxicity to the pancreas.
Treatments for pancreatitis are limited. Activation of transcription factor NF-kappaB, a key regulator of inflammatory molecule expression, is an early event in experimental pancreatitis and ...correlates with the inflammatory response. We report here that curcumin, a natural phytochemical known to inhibit NF-kappaB and activator protein (AP)-1, another important proinflammatory transcription factor, ameliorates pancreatitis in two rat models. In both cerulein pancreatitis and pancreatitis induced by a combination of ethanol diet and low-dose CCK, curcumin improved the severity of the disease as measured by a number of parameters (histology, serum amylase, pancreatic trypsin, and neutrophil infiltration). Curcumin markedly inhibited NF-kappaB and AP-1 activation, assessed by DNA binding and degradation of inhibitory IkappaB proteins, and the induction of mRNAs for cytokines IL-6 and TNF-alpha, the chemokine KC, and inducible nitric oxide synthase in pancreas. Curcumin also blocked CCK-induced NF-kappaB and AP-1 activation in isolated pancreatic acini. Our findings indicate that blocking key signals of the inflammatory response ameliorates pancreatitis in both ethanol and nonethanol models. They suggest that curcumin, which is currently in clinical trials for cancer prevention, may be useful for treatment of pancreatitis.
Citrofortunella microcarpa, locally known as calamansi in the Philippines, is an intergeneric hybrid between Citrus reticulata and Fortunella japonica. This fruit is widely cultivated in the ...Philippines for its fruit juice as an abundant source of vitamin C and as a condiment in many local foods in the country. Sadly, only the pulp is needed for squeezing while the peels are thrown after extracting the juice. Previous studies revealed that the peels of Citrus, as member of the Rutaceae family, can synthesize both coumarins and furanocoumarins wherein their derivates are used as oral anticoagulants which can inhibit vitamin K from functioning as a cofactor in the hepatic synthesis of the vitamin K-dependent coagulation factors II, VII, IX, and X. In this study, the extract of calamansi peelings were proven to have an anticoagulant property on blood samples from albino mice. This study will pave the way for scientists to allot time in studying calamansi peelings for it may be another source of medicine to help patients who are prone to have stroke, myocardial infarction, and other blood clotting diseases.
ABSTRACT
Severe necrotizing pancreatitis occurs in young female mice fed a choline‐deficient and ethionine‐supplemented (CDE) diet. Although the mechanism of the pancreatitis is unknown, one ...consequence of this diet is depletion of hepatic S‐adenosylmethionine (SAM). SAM formation is catalyzed by methionine adenosyltransferases (MATs), which are encoded by liver‐specific (MAT1A) and non‐liver‐specific (MAT2A) genes. In this work, we examined changes in pancreatic SAM homeostasis in mice receiving the CDE diet and the effect of SAM treatment. We found that both MAT forms are expressed in normal pancreas and pancreatic acini. After 48 h of the CDE diet, SAM levels decreased 50% and MAT1A‐encoded protein disappeared via post‐translational mechanisms, whereas MAT2A‐encoded protein increased via pretranslational mechanisms. CDE‐fed mice exhibited extensive necrosis, edema, and acute pancreatic inflammatory infiltration, which were prevented by SAM treatment. However, old female mice consuming the CDE diet that do not develop pancreatitis showed a similar fall in pancreatic SAM level. SAM was also protective in cerulein‐induced pancreatitis in the rat, but the protection was limited. Although the pancreatic SAM level fell by more than 80% in the MAT1A knockout mice, no pancreatitis developed. This study thus provides several novel findings. First, the so‐called liver‐specific MAT1A is highly expressed in the normal pancreas and pancreatic acini. Second, the CDE diet causes dramatic changes in the expression of MAT isozymes by different mechanisms. Third, in contrast to the situation in the liver, where absence of MAT1A and decreased hepatic SAM level can lead to spontaneous tissue injury, in the pancreas the roles of SAM and MAT1A appear more complex and remain to be defined.
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