M.D. Carretta

Andrographolide reduces IL-2 production in T-cells by interfering with NFAT and MAPK activation

The nuclear factor of activated T cells (NFAT) is a transcription factor essential for cytokine production during T-cell activation and is the target of several immunosuppressive drugs. Andrographolide is a diterpenic labdane that possesses anti-inflammatory and immunomodulatory effects. Several studies propose that andrographolide can reduce the immune response through inhibition of the nuclear factor kappa B (NF-kappaB) and mitogen-activated protein kinases (MAPK) such as extracellular signal regulated kinase 1/2 (ERK1/2) pathways. Moreover, andrographolide reduces IFN-gamma and IL-2 production induced by concanavalin A in murine T-cell. Nevertheless, the mechanisms involved in the decrease of cytokine production are unknown. In the present study, we determined that andrographolide reduced IL-2 production in Jurkat cells stimulated with phorbol myristate acetate and ionomycin (PMA/Ionomycin). We then showed that andrographolide reduced NFAT luciferase activity and interfered with its nuclear distribution, with these effects being linked to an increase in c-jun-N-terminal kinase (JNK) phosphorylation. Additionally, reduction of NF-kappaB activity in Jurkat cells treated with andrographolide was observed. Using Western blotting, we demonstrated that andrographolide decreased ERK1 and ERK5 phosphorylation induced by anti-CD3 or PMA/Ionomycin. Andrographolide did not affect cell viability at concentration of 10 and 50 muM; however, our results suggest that andrographolide increase early apoptosis at 100 muM. We concluded that andrographolide can exert immunomodulatory effects by interfering with NFAT activation and ERK1 and ERK5 phosphorylation in T-cells.

Store-operated calcium entry mediates intracellular alkalinization, ERK1/2, and Akt/PKB phosphorylation in bovine neutrophils

Neutrophil’s responses to G protein‐coupled chemoattractants are highly dependent on store‐operated calcium (Ca2+) entry (SOCE). Platelet‐activating factor (PAF), a primary chemoattractant, simultaneously increases cytosolic‐free Ca2+, intracellular pH (pHi), ERK1/2, and Akt/protein kinase B (PKB) phosphorylation. In this study, we looked at the efficacy of several putative SOCE inhibitors and whether SOCE mediates intracellular alkalinization, ERK1/2, and Akt/PKB phosphorylation in bovine neutrophils. We demonstrated that the absence of external Ca2+ and the presence of EGTA reduced the intracellular alkalinization and ERK1/2 phosphorylation induced by PAF, apparently via SOCE influx inhibition. Next, we tested the efficacy of several putative SOCE inhibitors such as 2‐aminoethoxydiphenyl borate (2‐APB), capsaicin, flufenamic acid, 1‐{β‐[3‐(4‐methoxy‐phenyl)propoxy]‐4‐methoxyphenethyl}‐1H‐imidazole hydrochloride (SK&F 96365), and N‐(4‐[3,5‐bis(trifluoromethyl)‐1H‐pyrazol‐1‐yl]phenyl)‐4‐methyl‐1,2,3‐thiadiazole‐5‐carboxamide (BTP2) on Ca2+ entry induced by PAF or thapsigargin. 2‐APB was the most potent SOCE inhibitor, followed by capsaicin and flufenamic acid. Conversely, SK&F 96365 reduced an intracellular calcium ([Ca2+]i) peak but SOCE partially. BTP2 did not show an inhibitory effect on [Ca2+]i following PAF stimuli. 2‐APB strongly reduced the pHi recovery, whereas the effect of flufenamic acid and SK&F 96365 was partial. Capsaicin and BTP2 did not affect the pHi changes induced by PAF. Finally, we observed that 2‐APB reduced the ERK1/2 and Akt phosphorylation completely, whereas the inhibition with flufenamic acid was partial. The results suggest that 2‐APB is the most potent SOCE inhibitor and support a key role of SOCE in pH alkalinization and PI‐3K–ERK1/2 pathway control. Finally, 2‐APB could be an important tool to characterize Ca2+ signaling in neutrophils.

Oleic acid induces intracellular calcium mobilization, MAPK phosphorylation, superoxide production and granule release in bovine neutrophils

Oleic acid (OA) is a nonesterified fatty acid that is released into the blood during lipomobilization at the time of calving in cows, a period where increased risk of infection and acute inflammation is observed. These data suggest potential OA-mediated regulation of innate immune responses. In the present study, we assessed the effects of OA on intracellular calcium release, ERK1/2 phosphorylation, superoxide production, CD11b expression and matrix metalloproteinase-9 (MMP-9) release in bovine neutrophils. Furthermore, the presence of GPR40, an OA receptor, was assessed by RT-PCR, immunoblotting and confocal microscopy. OA induced, in a dose-dependent manner, intracellular calcium mobilization, superoxide production and CD11b expression in bovine neutrophils; these effects were reduced by the intracellular chelating agent BAPTA-AM. OA also induced ERK2 phosphorylation and MMP-9 release. RT-PCR analysis detected mRNA expression of a bovine ortholog of the GPR40 receptor. Using a polyclonal antibody against human GPR40, we detected a protein of 31kDa by immunoblotting that was localized predominately in the plasma membrane. The selective agonist of GPR40, GW9508, induced intracellular calcium mobilization and ERK2 phosphorylation. In conclusion, OA can modulate bovine neutrophil responses in an intracellular calcium-dependent manner; furthermore, these responses could be induced by GPR40 activation.

2-Aminoethoxydiphenyl borate (2-APB) reduces respiratory burst, MMP-9 release and CD11b expression, and increases l-selectin shedding in bovine neutrophils

This study describes the effect of 2-aminoethoxydiphenyl borate (2-APB), a putative store-operated calcium (Ca(2+)) entry (SOCE) inhibitor, on reactive oxygen species (ROS) production, matrix metalloproteinase 9 (MMP-9) release, CD11b and l-selectin (CD62L) expression, size changes and apoptosis in bovine neutrophils stimulated with platelet-activating factor (PAF). It was observed that doses ⩾1μM 2-APB significantly reduced ROS production, whereas 50 and 100μM 2-APB reduced MMP-9 release induced by PAF. Moreover, concentrations ⩾10μM 2-APB reduced CD11b expression and increased l-selectin shedding. PAF induced size changes in neutrophils, and this effect was inhibited by 2-APB. From this work it is possible to conclude that 2-APB at concentrations that inhibit SOCE responses was able to inhibit ROS and MMP-9 release and CD11b expression, and increase l-selectin shedding, suggesting that the Ca(2+) channel involved in SOCE is a potential target for the development of new anti-inflammatory drugs in cattle.

Propionate induces the release of granules from bovine neutrophils

Short-chain fatty acids (SCFA) are produced by bacterial fermentation in the rumen of cattle and are the primary energy source in ruminants. Propionate is one of the main SCFA and it can exert multiple effects on the inflammatory process and neutrophil function via calcium (Ca(2+)) release, reactive oxygen species, and intracellular pH changes. However, currently no evidence has shown whether propionate can induce granule release from bovine neutrophils. The purpose of this study was to analyze the effect of propionate on granule release and to evaluate the expression of two G-protein coupled receptors-GPR41 and GPR43-that are activated by propionate. Neutrophil degranulation was assessed by quantifying the release of the neutrophil enzymes myeloperoxidase (MPO), lactoferrin, and matrix metalloprotease-9 (MMP-9) as markers of azurophil, specific granules, and gelatinase granules, respectively. Isolated bovine neutrophils were treated with millimolar concentrations of propionate (0.3, 3 and 30mM), and the cell-free supernatants were recovered. The stimulation of neutrophils with 0.3mM propionate induced the release of lactoferrin and MMP-9 as revealed by ELISA and gelatin zymography, respectively. Propionate at 30mM induced the release of MPO as demonstrated using an enzymatic assay. The role of intracellular Ca(2+) influx and the signaling pathways that may regulate the propionate effect on granules release were also determined. Reverse transcription (RT)-PCR and real-time PCR were performed to analyze the expression of GPR41 and GPR43 mRNA in bovine neutrophils. Both mRNA were detected, whereas the expression of GPR43 was higher than that of GPR41, and the synthetic agonists for this receptor, phenylacetamides 1 and 2, caused an increase in intracellular Ca(2+), lactoferrin, and MMP-9 release. These results support that propionate-induced granule release is mediated by intracellular Ca(2+) influx and activation of extracellular signal-regulated kinase ERK 1/2. We also propose a potential role of GPR43 in propionate-induced granule release from bovine neutrophils that may be involved in regulatory effects of propionate in the innate immune response in cattle.

Butyric acid stimulates bovine neutrophil functions and potentiates the effect of platelet activating factor

Increased short-chain fatty acid (SCFA) production is associated with subacute ruminal acidosis (SARA) and activation of inflammatory processes. In humans and rodents, SCFAs modulate inflammatory responses in the gut via free fatty acid receptor 2 (FFA2). In bovines, butyric acid is one of the most potent FFA2 agonists. Its expression in bovine neutrophils has recently been demonstrated, suggesting a role in innate immune response in cattle. This study aimed to evaluate if butyric acid modulates oxidative and non-oxidative functions or if it can potentiate other inflammatory mediators in bovine neutrophils. Our results showed that butyric acid can activate bovine neutrophils, inducing calcium (Ca(2+)) influx and mitogen-activated protein kinase (MAPK) phosphorylation, two second messengers involved in FFA2 activation. Ca(2+) influx induced by butyric acid was dependent on the extracellular and intracellular Ca(2+) source and phospholipase C (PLC) activation. Butyric acid alone had no significant effect on reactive oxygen species (ROS) production and chemotaxis; however, a priming effect on platelet-activating factor (PAF), a potent inflammatory mediator, was observed. Butyric acid increased CD63 expression and induced the release of neutrophil granule markers matrix metalloproteinase-9 (MMP-9) and lactoferrin. Finally, we observed that butyric acid induced neutrophil extracellular trap (NET) formation without affecting cellular viability. These findings suggest that butyric acid, a component of the ruminal fermentative process, can modulate the innate immune response of ruminants.

Differential free fatty acid receptor-1 (FFAR1/GPR40) signalling is associated with gene expression or gelatinase granule release in bovine neutrophils

Fatty acids have been recognized as regulators of immune function in addition to their known metabolic role. Long-chain fatty acids bind free fatty acid receptor (FFAR)-1/GPR40, which is expressed on bovine neutrophils, and increase responses such as granule release and gene expression. In this study, we investigated the molecular mechanisms governing the up-regulation of cyclooxygenase-2 (COX-2) and IL-8, as well as matrix metalloproteinase (MMP)-9 granule release in FFAR1/GPR40 agonist-stimulated neutrophils. Our results showed that natural (oleic and linoleic acid) and synthetic (GW9508) FFAR1/GPR40 agonists increased ERK1/2, p38 MAPK and Akt phosphorylation, and that the FFAR1/GPR40 antagonist GW1100 reduced these responses. We evaluated the levels of IκBα, a component of the classical activation pathway of the transcription factor NF-κB, and we observed IκBα reduction after stimulation with FFAR1/GPR40 agonists, an effect that was inhibited by GW1100 or the inhibitors UO126, SB203580 or LY294002. FFAR1/GPR40 agonists increased COX-2 and IL-8 expression, which was inhibited by GW1100 and an NF-κB inhibitor. Finally, the FFAR1/GPR40 agonist-induced MMP-9 granule release was reduced by GW1100 and UO126. In conclusion, FFAR1/GPR40 agonists differentially stimulate neutrophil functions; COX-2 and IL-8 are expressed after FFAR1/GPR40 activation via NF-κB, IκBα reduction is FFAR1/GPR40- and PI3K/MAPK-dependent, and MMP-9 granule release is FFAR1/GPR40- and ERK1/2-dependent.

fMLP-Induced IL-8 Release Is Dependent on NADPH Oxidase in Human Neutrophils

N-Formyl-methionyl-leucyl-phenylalanine (fMLP) and platelet-activating factor (PAF) induce similar intracellular signalling profiles; but only fMLP induces interleukin-8 (IL-8) release and nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase activity in neutrophils. Because the role of ROS on IL-8 release in neutrophils is until now controversial, we assessed if NADPH oxidase is involved in the IL-8 secretions and PI3K/Akt, MAPK, and NF-κB pathways activity induced by fMLP. Neutrophils were obtained from healthy volunteers. IL-8 was measured by ELISA, IL-8 mRNA by qPCR, and ROS production by luminol-amplified chemiluminescence, reduction of ferricytochrome c, and FACS. Intracellular pH changes were detected by spectrofluorescence. ERK1/2, p38 MAPK, and Akt phosphorylation were analysed by immunoblotting and NF-κB was analysed by immunocytochemistry. Hydroxy-3-methoxyaceto-phenone (HMAP), diphenyleneiodonium (DPI), and siRNA Nox2 reduced the ROS and IL-8 release in neutrophils treated with fMLP. HMAP, DPI, and amiloride (a Na+/H+ exchanger inhibitor) inhibited the Akt phosphorylation and did not affect the p38 MAPK and ERK1/2 activity. DPI and HMAP reduced NF-κB translocation induced by fMLP. We showed that IL-8 release induced by fMLP is dependent on NADPH oxidase, and ROS could play a redundant role in cell signalling, ultimately activating the PI3K/Akt and NF-κB pathways in neutrophils.

2-Aminoethoxydiphenyl borate (2-APB) reduces alkaline phosphatase release, CD63 expression, F-actin polymerization and chemotaxis without affecting the phagocytosis activity in bovine neutrophils.

2-Aminoethoxydiphenyl borate (2-APB) interferes with the Ca(2+) influx and reduces the ROS production, gelatinase secretion and CD11b expression in bovine neutrophils. Moreover, it has been suggested that inhibition of the Ca(2+) channel involved in the store operated Ca(2+) entry (SOCE) is a potential target for the development of new anti-inflammatory drugs in cattle, however it is unknown whether 2-APB affects neutrophil functions associated with the innate immune response. This study describes the effect of 2-APB, a putative SOCE inhibitor, on alkaline phosphatase activity a marker of secretory vesicles, CD63 a marker for azurophil granules, F-actin polymerization and in vitro chemotaxis in bovine neutrophils stimulated with platelet-activating factor (PAF). Also, we evaluated the effect of 2-APB in the phagocytic activity against Escherichia coli and Staphylococcus aureus bioparticles. We observed that doses of 2-APB ≥10 μM significantly reduced alkaline phosphatase activity and in vitro chemotaxis, whereas concentrations of 2-APB ≥50 μM reduced CD63 expression and F-actin polymerization. Finally, we observed that 2-APB did not affect the phagocytic activity in neutrophils incubated with E. coli and S. aureus bioparticles. We concluded that inhibition of Ca(2+) influx could be a useful strategy to reduce inflammatory process in cattle.

Oxidative response of neutrophils to platelet-activating factor is altered during acute ruminal acidosis induced by oligofructose in heifers

Reactive oxygen species (ROS) production is one of the main mechanisms used to kill microbes during innate immune response. D-lactic acid, which is augmented during acute ruminal acidosis, reduces platelet activating factor (PAF)-induced ROS production and L-selectin shedding in bovine neutrophils in vitro. This study was conducted to investigate whether acute ruminal acidosis induced by acute oligofructose overload in heifers interferes with ROS production and L-selectin shedding in blood neutrophils. Blood neutrophils and plasma were obtained by jugular venipuncture, while ruminal samples were collected using rumenocentesis. Lactic acid from plasma and ruminal samples was measured by HPLC. PAF-induced ROS production and L-selectin shedding were measured in vitro in bovine neutrophils by a luminol chemiluminescence assay and flow cytometry, respectively. A significant increase in ruminal and plasma lactic acid was recorded in these animals. Specifically, a decrease in PAF-induced ROS production was observed 8 h after oligofructose overload, and this was sustained until 48 h post oligofructose overload. A reduction in PAF-induced L-selectin shedding was observed at 16 h and 32 h post oligofructose overload. Overall, the results indicated that neutrophil PAF responses were altered in heifers with ruminal acidosis, suggesting a potential dysfunction of the innate immune response.