The Role of the Pathogen Dose and PI3Kγ in Immunometabolic Reprogramming of Microglia for Innate Immune Memory.

Microglia, the innate immune cells of the CNS, exhibit long-term response changes indicative of innate immune memory (IIM). Our previous studies revealed IIM patterns of microglia with opposing immune phenotypes: trained immunity after a low dose and immune tolerance after a high dose challenge with pathogen-associated molecular patterns (PAMP). Compelling evidence shows that innate immune cells adopt features of IIM via immunometabolic control. However, immunometabolic reprogramming involved in the regulation of IIM in microglia has not been fully addressed. Here, we evaluated the impact of dose-dependent microglial priming with ultra-low (ULP, 1 fg/mL) and high (HP, 100 ng/mL) lipopolysaccharide (LPS) doses on immunometabolic rewiring. Furthermore, we addressed the role of PI3Kγ on immunometabolic control using naïve primary microglia derived from newborn wild-type mice, PI3Kγ-deficient mice and mice carrying a targeted mutation causing loss of lipid kinase activity. We found that ULP-induced IIM triggered an enhancement of oxygen consumption and ATP production. In contrast, HP was followed by suppressed oxygen consumption and glycolytic activity indicative of immune tolerance. PI3Kγ inhibited glycolysis due to modulation of cAMP-dependent pathways. However, no impact of specific PI3Kγ signaling on immunometabolic rewiring due to dose-dependent LPS priming was detected. In conclusion, immunometabolic reprogramming of microglia is involved in IIM in a dose-dependent manner via the glycolytic pathway, oxygen consumption and ATP production: ULP (ultra-low-dose priming) increases it, while HP reduces it.

The abnormal function of CD39+ regulatory T cells could be corrected by high-dose dexamethasone in patients with primary immune thrombocytopenia.

Primary immune thrombocytopenia is an autoimmune disease, characterized with decreased platelet and increased risk of bleeding. Recent studies have shown the reduction and dysfunction of regulatory T (Treg) cells in ITP patients. CD39 is highly expressed on the surface of Treg cells. It degrades ATP to AMP and CD73 dephosphorylates AMP into adenosine. Then adenosine binds with adenosine receptor and suppresses immune response by activating Treg cells and inhibiting the release of inflammatory cytokines from effector T (Teff) cells. Adenosine receptor has several subtypes and adenosine A2A receptor (A2AR) plays a crucial role especially within lymphocytes. The CD39+ Treg cells and the expression of A2AR showed abnormality in some autoimmune disease. But knowledge of CD39+ Treg cells and A2AR which are crucial in the adenosine immunosuppressive pathway is still limited in ITP. Thirty-one adult patients with newly diagnosed ITP were enrolled in this study. CD39 and A2AR expression was measured by flow cytometry and RT-PCR. The function of CD39 was reflected by the change of ATP concentration detected by CellTiter-Glo Luminescent Cell Viability Assay. CD39 expression within CD4+CD25+ Treg cells in ITP patients was decreased compared to normal controls. After high-dose dexamethasone therapy, response (R) group showed increased CD39 expression within Treg cells while non-response (NR) group did not show any difference in contrast to those before treatment. The expression of A2AR in CD4+CD25- Teff and CD4+CD25+ Treg cells was both lower in ITP patients than that of normal controls. After therapy, CD4+CD25- Teff cells had higher A2AR expression while CD4+CD25+ Treg cells did not show any difference in comparison to that before treatment. The enzymatic activity of CD39 was damaged in ITP patients and improved after high-dose dexamethasone therapy. In ITP, there was not only numerical decrease but also impaired enzymatic activity in CD39+ Treg cells. After high-dose dexamethasone treatment, these two defects could be reversed. Our results also suggested that ITP patients had reduced A2AR expression in both CD4+CD25+ Treg cells and CD4+CD25- Teff cells. CD4+CD25- Teff cells had increased A2AR expression after treatment.

ATP redirects cytokine trafficking and promotes novel membrane TNF signaling via microvesicles.

Cellular stress or injury induces release of endogenous danger signals such as ATP, which plays a central role in activating immune cells. ATP is essential for the release of nonclassically secreted cytokines such as IL-1ß but, paradoxically, has been reported to inhibit the release of classically secreted cytokines such as TNF. Here, we reveal that ATP does switch off soluble TNF (17 kDa) release from LPS-treated macrophages, but rather than inhibiting the entire TNF secretion, ATP packages membrane TNF (26 kDa) within microvesicles (MVs). Secretion of membrane TNF within MVs bypasses the conventional endoplasmic reticulum- and Golgi transport-dependent pathway and is mediated by acid sphingomyelinase. These membrane TNF-carrying MVs are biologically more potent than soluble TNF in vivo, producing significant lung inflammation in mice. Thus, ATP critically alters TNF trafficking and secretion from macrophages, inducing novel unconventional membrane TNF signaling via MVs without direct cell-to-cell contact. These data have crucial implications for this key cytokine, particularly when therapeutically targeting TNF in acute inflammatory diseases.-Soni, S., O'Dea, K. P., Tan, Y. Y., Cho, K., Abe, E., Romano, R., Cui, J., Ma, D., Sarathchandra, P., Wilson, M. R., Takata, M. ATP redirects cytokine trafficking and promotes novel membrane TNF signaling via microvesicles.

Autophagy Mediates Interleukin-1ß Secretion in Human Neutrophils.

Interleukin-1ß (IL-1ß), a major pro-inflammatory cytokine, is a leaderless cytosolic protein whose secretion does not follow the classical endoplasmic reticulum-to-Golgi pathway, and for which a canonical mechanism of secretion remains to be established. Neutrophils are essential players against bacterial and fungi infections. These cells are rapidly and massively recruited from the circulation into infected tissues and, beyond of displaying an impressive arsenal of toxic weapons effective to kill pathogens, are also an important source of IL-1ß in infectious conditions. Here, we analyzed if an unconventional secretory autophagy mechanism is involved in the exportation of IL-1ß by these cells. Our findings indicated that inhibition of autophagy with 3-methyladenine and Wortmannin markedly reduced IL-1ß secretion induced by LPS + ATP, as did the disruption of the autophagic flux with Bafilomycin A1 and E64d. These compounds did not noticeable affect neutrophil viability ruling out that the effects on IL-1ß secretion were due to cell death. Furthermore, VPS34IN-1, a specific autophagy inhibitor, was still able to reduce IL-1ß secretion when added after it was synthesized. Moreover, siRNA-mediated knockdown of ATG5 markedly reduced IL-1ß secretion in neutrophil-differentiated PLB985 cells. Upon LPS + ATP stimulation, IL-1ß was incorporated to an autophagic compartment, as was revealed by its colocalization with LC3B by confocal microscopy. Overlapping of IL-1ß-LC3B in a vesicular compartment peaked before IL-1ß increased in culture supernatants. On the other hand, stimulation of autophagy by cell starvation augmented the colocalization of IL-1ß and LC3B and then promoted neutrophil IL-1ß secretion. In addition, specific ELISAs indicated that although both IL-1ß and pro-IL-1ß are released to culture supernatants upon neutrophil stimulation, autophagy only promotes IL-1ß secretion. Furthermore, the serine proteases inhibitor AEBSF reduced IL-1ß secretion. Moreover, IL-1ß could be also found colocalizing with elastase, suggesting both some vesicles containing IL-1ß intersect azurophil granules content and that serine proteases also regulate IL-1ß secretion. Altogether, our findings indicate that an unconventional autophagy-mediated secretory pathway mediates IL-1ß secretion in human neutrophils.

Extracellular ATP and adenosine: The Yin and Yang in immune responses?

Extracellular adenosine 5'-triphosphate (ATP) and adenosine molecules are intimately involved in immune responses. ATP is mostly a pro-inflammatory molecule and is released during hypoxic condition and by necrotic cells, as well as by activated immune cells and endothelial cells. However, under certain conditions, for instance at low concentrations or at prolonged exposure, ATP may also have anti-inflammatory properties. Extracellular ATP can activate both P2X and P2Y purinergic receptors. Extracellular ATP can be hydrolyzed into adenosine in a two-step enzymatic process involving the ectonucleotidases CD39 (ecto-apyrase) and CD73. These enzymes are expressed by many cell types, including endothelial cells and immune cells. The counterpart of ATP is adenosine, which is produced by breakdown of intra- or extracellular ATP. Adenosine has mainly anti-inflammatory effects by binding to the adenosine, or P1, receptors (A1, A2A, A2B, and A3). These receptors are also expressed in many cells, including immune cells. The final effect of ATP and adenosine in immune responses depends on the fine regulatory balance between the 2 molecules. In the present review, we will discuss the current knowledge on the role of these 2 molecules in the immune responses.

Activation of Peptidylarginine Deiminase in the Salivary Glands of Balb/c Mice Drives the Citrullination of Ro and La Ribonucleoproteins.

The goal of the present study was to determine whether peptidylarginine deiminase PAD2 and PAD4 enzymes are present in Balb/c mouse salivary glands and whether they are able to citrullinate Ro and La ribonucleoproteins. Salivary glands from Balb/c mice were cultured in DMEM and supplemented with one of the following stimulants: ATP, LPS, TNF, IFNγ, or IL-6. A control group without stimulant was also evaluated. PAD2, PAD4, citrullinated peptides, Ro60, and La were detected by immunohistochemistry and double immunofluorescence. PAD2 and PAD4 mRNAs and protein expression were detected by qPCR and Western blot analysis. PAD activity was assessed using an antigen capture enzyme-linked immunosorbent assay. LPS, ATP, and TNF triggered PAD2 and PAD4 expression; in contrast, no expression was detected in the control group (p < 0.001). PAD transcription slightly increased in response to stimulation. Additionally, PAD2/4 activity modified the arginine residues of a reporter protein (fibrinogen) in vitro. PADs citrullinated Ro60 and La ribonucleoproteins in vivo. Molecular stimulants induced apoptosis in ductal cells and the externalization of Ro60 and La ribonucleoproteins onto apoptotic membranes. PAD enzymes citrullinate Ro and La ribonucleoproteins, and this experimental approach may facilitate our understanding of the role of posttranslational modifications in the pathophysiology of Sjögren's syndrome.

Enhancement of Muscle T Regulatory Cells and Improvement of Muscular Dystrophic Process in mdx Mice by Blockade of Extracellular ATP/P2X Axis.

Infiltration of immune cells and chronic inflammation substantially affect skeletal and cardiac muscle degeneration in Duchenne muscular dystrophy. In the immune system, extracellular adenosine triphosphate (ATP) released by dying cells is sensed as a danger associated molecular pattern through P2 purinergic receptors. Specifically, the P2X7 subtype has a prominent role in regulating immune system physiology and contributes to inflammasome activation also in muscle cells. Here, we show that in vivo blockade of the extracellular ATP/P2X purinergic signaling pathway by periodate-oxidized ATP delayed the progression of the dystrophic phenotype and dampened the local inflammatory response in mdx mice, a spontaneous mouse model of dystrophin deficiency. Reduced infiltration of leukocytes and macrophages and decreased expression of IL-6 were revealed in the muscles of periodate-oxidized ATP-treated mdx mice. Concomitantly, an increase in Foxp3(+) immunosuppressive regulatory T cells was observed and correlated with enhanced myofiber regeneration. Moreover, we detected reduced concentrations of profibrotic cytokines, including transforming growth factor-ß and connective tissue growth factor, in muscles of periodate-oxidized ATP-treated mdx mice. The improvement of inflammatory features was associated with increased strength and reduced necrosis, thus suggesting that pharmacologic purinergic antagonism altering the adaptive immune component in the muscle infiltrates might represent a promising therapeutic approach in Duchenne muscular dystrophy.

Inflammasome-dependent and -independent IL-18 production mediates immunity to the ISCOMATRIX adjuvant.

Adjuvants are an essential component of modern vaccines and used for their ability to elicit immunity to coadministered Ags. Many adjuvants in clinical development are particulates, but how they drive innate and adaptive immune responses remains poorly understood. Studies have shown that a number of vaccine adjuvants activate inflammasome pathways in isolated APCs. However, the contribution of inflammasome activation to vaccine-mediated immunity in vivo remains controversial. In this study, we evaluated immune cell responses to the ISCOMATRIX adjuvant (IMX) in mice. Like other particulate vaccine adjuvants, IMX potently activated the NALP-3-ASC-Caspase-1 inflammasome in APCs, leading to IL-1ß and IL-18 production. The IL-18R pathway, but not IL-1R, was required for early innate and subsequent cellular immune responses to a model IMX vaccine. APCs directly exposed to IMX underwent an endosome-mediated cell-death response, which we propose initiates inflammatory events locally at the injection site. Importantly, both inflammasome-related and -unrelated pathways contributed to IL-18 dependence in vivo following IMX administration. TNF-α provided a physiological priming signal for inflammasome-dependent IL-18 production by APCs, which correlated with reduced vaccine-mediated immune cell responses in TNF-α- or TNFR-deficient mice. Taken together, our findings highlight an important disconnect between the mechanisms of vaccine adjuvant action in vitro versus in vivo.

PAMPs and DAMPs in allergy exacerbation models.

Sensitization of mice to real-life allergens or harmless antigen with the use of adjuvants will lead to the induction of DAMPs in the immune system. We have shown that the Th2-inducing adjuvant aluminum hydroxide or exposure of the airways to house dust mite leads to the release of DAMPs: uric acid, ATP, and IL-1. Exposure to DAMPs or PAMPs present in allergens or added to harmless allergens, such as the experimental allergen ovalbumin, induces several immune responses, including cellular influx and activation. Cellular influx can be analyzed by flow cytometry. Likewise, cellular activation can be assessed by measuring increased expression and release of chemokines and cytokines. These inflammatory mediators can be analyzed by ELISA or confocal microscopy. Here, we describe the protocols for these assessments and a protocol that takes advantage of bone marrow chimeric mice to further elucidate mechanism.

Nitric oxide affects immune cells bioenergetics: long-term effects of nitric-oxide derivatives on leukaemic Jurkat cell metabolism.

Major advances in dissecting mechanisms of NO-induced down-regulation of the anti-tumour specific T-cell function have been accomplished during the last decade. In this work, we studied the effects of a NO donor (AT38) on leukaemic Jurkat cell bioenergetics. Culturing Jurkat cells in the presence of AT38 triggered irreversible inhibition of cell respiration, led to the depletion of 50% of the intracellular ATP content and induced the arrest of cell proliferation and the loss of cell viability. Although a deterioration of the overall metabolic activity has been observed, glycolysis was stimulated, as revealed by the increase of glucose uptake and lactate accumulation rates as well as by the up-regulation of GLUT-1 and PFK-1 mRNA levels. In the presence of NO, cell ATP was rapidly consumed by energy-requiring apoptosis mechanisms; under a glucose concentration of about 12.7mM, cell death was switched from apoptosis into necrosis. Exposure of Jurkat cells to DMSO (1%, v/v), SA and AT55, the non-NO releasing moiety of AT38, failed to modulate neither cell proliferation nor bioenergetics. Thus, as for all NSAIDs, beneficial effects of AT38 on tumour regression are accompanied by the suppression of the immune system. We then showed that pre-treating Jurkat cells with low concentration of cyclosporine A, a blocker of the mitochondrial transition pore, attenuates AT38-induced inhibition of cell proliferation and suppresses cell death. Finally, we have studied and compared the effects of nitrite and nitrate on Jurkat cells to those of NO and we are providing evidence that nitrate, which is considered as a biologically inert anion, has a concentration and time-dependent immunosuppressive potential.

Augmentation of cutaneous immune responses by ATP gamma S: purinergic agonists define a novel class of immunologic adjuvants.

Extracellular nucleotides activate ligand-gated P2XR ion channels and G protein-coupled P2YRs. In this study we report that intradermal administration of ATPgammaS, a hydrolysis-resistant P2 agonist, results in an enhanced contact hypersensitivity response in mice. Furthermore, ATPgammaS enhanced the induction of delayed-type hypersensitivity to a model tumor vaccine in mice and enhanced the Ag-presenting function of Langerhans cells (LCs) in vitro. Exposure of a LC-like cell line to ATPgammaS in the presence of LPS and GM-CSF augmented the induction of I-A, CD80, CD86, IL-1beta, and IL-12 p40 while inhibiting the expression of IL-10, suggesting that the immunostimulatory activities of purinergic agonists in the skin are mediated at least in part by P2Rs on APCs. In this regard, an LC-like cell line was found to express mRNA for P2X(1), P2X(7), P2Y(1), P2Y(2), P2Y(4), P2Y(9), and P2Y(11) receptors. We suggest that ATP, when released after trauma or infection, may act as an endogenous adjuvant to enhance the immune response, and that P2 agonists may augment the efficacy of vaccines.

Limited ATP generation in cells of mycobacterium leprae thai-53 strain in enriched kirchner liquid medium containing adenosine

The ATP generation in cells of Mycobacterium leprae Thai-53 strain takes place in vitro when the cells are cultivated in Kirchner liquid medium, pH 7.0, enriched with egg-yolk solution, pyruvate, transferrin, and adenosine at 30 degrees C. Among the supplements, adenosine was key and critical for the ATP generation. The optimal concentration of adenosine was 50 micrograms/ml of the medium. ATP generation, however, was limited; the rates of increase in ATP content extracted from the cells were approximately two- to threefold compared to that of the starting samples, and the increase reached a maximum at 4 or 6 weeks after incubation. No significant ATP generation in M. leprae cells was demonstrated in medium at pH 6.2 or pH 6.6, in the original Kirchner medium with or without adenosine, or when cultured at 37 degrees C, or when containing an antileprosy drug. No detectable increase in the number of M. leprae cells was observed with the increase in intracellular ATP content and DNA replication. No effect was seen with renewal of the cultured medium by freshly prepared medium at 6 weeks' cultivation on the progressive ATP generation in M. leprae.

Extracellular ATP and TNF-alpha synergize in the activation and maturation of human dendritic cells.

Extracellular ATP mediates numerous biological activities by interacting with plasma membrane P2 purinergic receptors. Recently, P2 receptors have been described on dendritic cells (DC), but their functional role remains unclear. Proposed functions include improved Ag presentation, cytokine production, chemotaxis, and induction of apoptosis. We investigated the effects of ATP and of other P2 receptor agonists on endocytosis, phenotype, IL-12 secretion, and T cell stimulatory capacity of human monocyte-derived DC. We found that in the presence of extracellular ATP, DC transiently increase their endocytotic activity. Subsequently, DC up-regulate CD86, CD54, and MHC-II; secrete IL-12; and exhibit an improved stimulatory capacity for allogeneic T cells. These effects were more pronounced when chemically modified ATP derivatives with agonistic activity on P2 receptors, which are resistent to degradation by ectonucleotidases, were applied. Furthermore, ATP and TNF-alpha synergized in the activation of DC. Stimulated with a combination of ATP and TNF-alpha, DC expressed the maturation marker CD83, secreted large amounts of IL-12, and were potent stimulators of T cells. In the presence of the P2 receptor antagonist suramin, the effects of ATP were completely abolished. Our results suggest that extracellular ATP may play an important immunomodulatory role by activating DC and by skewing the immune reaction toward a Th1 response through the induction of IL-12 secretion.