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In Vitro Experimental Model of Trained Innate Immunity in Human Primary Monocytes.

ABSTRACT: Innate immune memory, or trained immunity, has recently been described to be an important property of cells of the innate immune system. Due to the increased interest in this important new field of immunological investigation, we sought to determine the optimal conditions for an in vitro experimental protocol of monocyte training using three of the most commonly used training stimuli from the literature: ?-glucan, the bacillus Calmette-Guérin (BCG) vaccine, and oxidized low-density lipoprotein (oxLDL). We investigated and optimized a protocol of monocyte trained immunity induced by an initial training period with ?-glucan, BCG, or oxLDL, followed by washing and resting of the cells and, thereafter, restimulation with secondary bacterial stimuli. The training and resting time intervals were varied to identify the optimal setting for the long-term induction of trained immunity. Trained immunity was assessed in terms of the secondary cytokine response, the production of reactive oxygen species, cell morphology, and induction of glycolysis. Monocytes primed with ?-glucan, BCG, and oxLDL showed increased pro- and anti-inflammatory cytokine responses upon restimulation with nonrelated stimuli. Also, all three stimuli induced a switch to glycolysis (the Warburg effect). These effects were most pronounced when the training interval was 24 h and the resting time interval was 6 days. Training with BCG and oxLDL also led to the increased production of reactive oxygen species, whereas training with ?-glucan led to the decreased production of reactive oxygen species. We describe the optimal conditions for an in vitro experimental model with human primary monocytes for study of the induction of trained innate immunity by microbial and metabolic stimuli.

SUBMITTER: Bekkering S

PROVIDER: S-EPMC5139603 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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In Vitro Experimental Model of Trained Innate Immunity in Human Primary Monocytes.

Bekkering Siroon S Blok Bastiaan A BA Joosten Leo A B LA Riksen Niels P NP van Crevel Reinout R Netea Mihai G MG

Clinical and vaccine immunology : CVI 20161205 12

Innate immune memory, or trained immunity, has recently been described to be an important property of cells of the innate immune system. Due to the increased interest in this important new field of immunological investigation, we sought to determine the optimal conditions for an in vitro experimental protocol of monocyte training using three of the most commonly used training stimuli from the literature: β-glucan, the bacillus Calmette-Guérin (BCG) vaccine, and oxidized low-density lipoprotein ( ...[more]

PMID: 27733422

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Project description:Introduction: Cells of the innate immune system particularly monocytes and macrophages have been recognized as pivotal players both during the initial insult as well as the chronic phase of atherosclerosis. It has recently been shown that oxidized low-density lipoprotein (oxLDL) induces a long-term pro-inflammatory response in monocytes due to epigenetic and metabolic reprogramming, an emerging new concept called trained innate immunity. Changes in the cellular redox state are crucial events in the regulation of many physiologic functions in macrophages including transcription, differentiation and inflammatory response. Here we have analyzed the role of reactive oxygen species (ROS) in regulating this proinflammatory monocyte priming in response to oxLDL-treatment. Methods and Results: Human monocytes were isolated and incubated with oxLDL for 24 h. After 5 days of resting, oxLDL treated cells produced significantly more inflammatory cytokines upon restimulation with the TLR2-agonist Pam3cys. Furthermore, oxLDL incubation induced persistent mTOR activation, ROS formation, HIF1? accumulation and HIF1? target gene expression, while pharmacologic mTOR inhibition or siRNA mediated inhibition of the mTORC1 subunit Raptor prevented ROS formation and proinflammatory priming. mTOR dependent ROS formation was associated with increased expression of NAPDH oxidases and necessary for the emergence of the primed phenotype as antioxidant treatment blocked oxLDL priming. Inhibition of cytosolic ROS formation could also block mTOR activation and HIF1? accumulation suggesting a positive feedback loop between mTOR and cytosolic ROS. Although mitochondrial ROS scavenging did not block HIF1?-accumulation at an early time point (24 h), it was persistently reduced on day 6. Therefore, mitochondrial ROS formation appears to occur initially downstream of the mTOR-cytoROS-HIF1? feedback loop but seems to be a crucial factor that controls the long-term activation of the mTOR-HIF1?-axis. Conclusion: In summary, our data demonstrate that mTOR dependent ROS production controls the oxLDL-induced trained innate immunity phenotype in human monocyte derived macrophages. Pharmacologic modulation of these pathways might provide a potential approach to modulate inflammation, associated with aberrant monocyte activation, during atherosclerosis development.

Project description:BackgroundNewborns exhibit distinct immune responses and are at high risk of infection. Neonatal immunization with BCG, the live attenuated vaccine against tuberculosis (TB), is associated with broad protection against a range of unrelated pathogens, possibly reflecting vaccine-induced training of innate immune cells ("innate memory"). However, little is known regarding the impact of age on BCG-induced innate responses.ObjectiveEstablish an age-specific human monocyte in vitro training platform to characterize and compare BCG-induced primary and memory cytokine responses and immunometabolic shifts.Design/methodsHuman neonatal and adult CD33-selected monocytes were stimulated for 24h with RPMI (control) or BCG (Danish strain) in 10% autologous serum, washed and cultured for 5 additional days, prior to re-stimulation with the TLR4 agonist LPS for another 24h. Supernatants were collected at Day 1 (D1) to measure primary innate responses and at Day 7 (D7) to assess memory innate responses by ELISA and multiplex cytokine and chemokine assays. Lactate, a signature metabolite increased during trained immunity, was measured by colorimetric assay.ResultsCytokine production by human monocytes differed significantly by age at D1 (primary, BCG 1:750 and 1:100 vol/vol, p<0.0001) and D7 (innate memory response, BCG 1:100 vol/vol, p<0.05). Compared to RPMI control, newborn monocytes demonstrated greater TNF (1:100, 1:10 vol/vol, p<0.01) and IL-12p40 (1:100 vol/vol, p<0.05) production than adult monocytes (1:100, p<0.05). At D7, while BCG-trained adult monocytes, as previously reported, demonstrated enhanced LPS-induced TNF production, BCG-trained newborn monocytes demonstrated tolerization, as evidenced by significantly diminished subsequent LPS-induced TNF (RPMI vs. BCG 1:10, p <0.01), IL-10 and CCL5 production (p<0.05). With the exception of IL-1RA production by newborn monocytes, BCG-induced monocyte production of D1 cytokines/chemokines was inversely correlated with D7 LPS-induced TNF in both age groups (p<0.0001). Compared to BCG-trained adult monocytes, newborn monocytes demonstrated markedly impaired BCG-induced production of lactate, a metabolite implicated in immune training in adults.ConclusionsBCG-induced human monocyte primary- and memory-innate cytokine responses were age-dependent and accompanied by distinct immunometabolic shifts that impact both glycolysis and training. Our results suggest that immune ontogeny may shape innate responses to live attenuated vaccines, suggesting age-specific approaches to leverage innate training for broad protection against infection.

Project description:Objective: Damage and pathogen associated molecular patterns such as oxidized low-density lipoprotein (oxLDL) or bacillus Calmette-Guerin (BCG) vaccine can induce long term pro-inflammatory priming in monocytes and macrophages due to metabolic and epigenetic reprogramming-an emerging new concept called trained innate immunity. Vascular smooth muscle cells express pattern recognition receptors involved in trained innate immunity in monocytes. Here we investigated whether the mechanisms of trained innate immunity also control a proinflammatory phenotype in human coronary smooth muscle cells. Methods: Human coronary smooth muscle cells were primed with oxLDL or BCG for 24 h. After a resting time of 4 to 7 days, the cells were restimulated with either PAM3cys4, LPS or TNF? and cytokine production or mRNA expression were measured. Then, mechanisms of monocyte trained innate immunity were analyzed in smooth muscle cells, including receptors, intracellular pathways as well as metabolic and epigenetic reprogramming. Results: Priming with oxLDL or BCG lead to a significantly increased production of IL6, IL8 and MCP-1 following restimulation. OxLDL priming had little effect on the expression of macrophage or SMC marker genes. Proinflammatory priming of smooth muscle cells induced mTOR-HIF1?-signaling and could be blocked by mTOR-, TLR2-, and TLR4-inhibition. Finally, metabolic and epigenetic mechanisms of trained innate immunity in monocytes could be replicated in smooth muscle cells, including increased glucose consumption, lactate production, responsiveness to 6-fluoromevalonate and mevalonate treatment and inhibition of priming by the histone methyltransferase inhibitor methylthioadenosine (MTA). Conclusion: We demonstrate for the first time that mechanisms of the so called trained innate immunity control a proinflammatory phenotype in non-immune cells of the vascular wall. Our findings warrant further research into the specificity of trained innate immunity as an immune cell response as well as the mechanisms of vascular smooth muscle cells inflammation.

Project description:Antimicrobial resistance (AMR) is a significant problem in health care, animal health, and food safety. To limit AMR, there is a need for alternatives to antibiotics to enhance disease resistance and support judicious antibiotic usage in animals and humans. Immunomodulation is a promising strategy to enhance disease resistance without antibiotics in food animals. One rapidly evolving field of immunomodulation is innate memory in which innate immune cells undergo epigenetic changes of chromatin remodeling and metabolic reprogramming upon a priming event that results in either enhanced or suppressed responsiveness to secondary stimuli (training or tolerance, respectively). Exposure to live agents such as bacille Calmette-Guerin (BCG) or microbe-derived products such as LPS or yeast cell wall ß-glucans can reprogram or "train" the innate immune system. Over the last decade, significant advancements increased our understanding of innate training in humans and rodent models, and strategies are being developed to specifically target or regulate innate memory. In veterinary species, the concept of enhancing the innate immune system is not new; however, there are few available studies which have purposefully investigated innate training as it has been defined in human literature. The development of targeted approaches to engage innate training in food animals, with the practical goal of enhancing the capacity to limit disease without the use of antibiotics, is an area which deserves attention. In this review, we provide an overview of innate immunomodulation and memory, and the mechanisms which regulate this long-term functional reprogramming in other animals (e.g., humans, rodents). We focus on studies describing innate training, or similar phenomenon (often referred to as heterologous or non-specific protection), in cattle, sheep, goats, swine, poultry, and fish species; and discuss the potential benefits and shortcomings of engaging innate training for enhancing disease resistance.

Dataset Information

In Vitro Experimental Model of Trained Innate Immunity in Human Primary Monocytes.

Publications

In Vitro Experimental Model of Trained Innate Immunity in Human Primary Monocytes.

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