Exogenous Vitamin D3 Modulates Response of Bovine Macrophages to Mycobacterium avium subsp. paratuberculosis Infection and Is Dependent Upon Stage of Johne’s Disease

Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of ruminant enteritis, targets intestinal macrophages. During infection, macrophages contribute to mucosal inflammation and development of granulomas in the small intestine which worsens as disease progression occurs. Vitamin D3 is an immunomodulatory steroid hormone with beneficial roles in host-pathogen interactions. Few studies have investigated immunologic roles of 25-hydroxyvitamin D3 (25(OH)D3) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in cattle, particularly cattle infected with MAP. This study examined the effects of exogenous vitamin D3 on immune responses of monocyte derived macrophages (MDMs) isolated from dairy cattle naturally infected with MAP. MDMs were pre-treated with ± 100 ng/ml 25(OH)D3 or ± 4 ng/ml 1,25(OH)2D3, then incubated 24 hrs with live MAP in the presence of their respective pre-treatment concentrations. Following treatment with either vitamin D3 analog, phagocytosis of MAP by MDMs was significantly greater in clinically infected animals, with a greater amount of live and dead bacteria. Clinical cows had significantly less CD40 surface expression on MDMs compared to subclinical cows and noninfected controls. 1,25(OH)2D3 also significantly increased nitrite production in MAP infected cows. 1,25(OH)2D3 treatment played a key role in upregulating secretion of pro-inflammatory cytokines IL-1β and IL-12 while downregulating IL-10, IL-6, and IFN-γ. 1,25(OH)2D3 also negatively regulated transcripts of CYP24A1, CYP27B1, DEFB7, NOS2, and IL10. Results from this study demonstrate that vitamin D3 compounds, but mainly 1,25(OH)2D3, modulate both pro- and anti-inflammatory immune responses in dairy cattle infected with MAP, impacting the bacterial viability within the macrophage.

Enantioselective Synthesis and Pharmacological Evaluation of Aza-CGP37157–Lipoic Acid Hybrids for the Treatment of Alzheimer’s Disease

Hybrids based on an aza-analogue of CGP37157, a mitochondrial Na+/Ca2+ exchanger antagonist, and lipoic acid were obtained in order to combine in a single molecule the antioxidant and NRF2 induction properties of lipoic acid and the neuroprotective activity of CGP37157. The four possible enantiomers of the hybrid structure were synthesized by using as the key step a fully diastereoselective reduction induced by Ellman’s chiral auxiliary. After computational druggability studies that predicted good ADME profiles and blood–brain permeation for all compounds, the DPPH assay showed moderate oxidant scavenger capacity. Following a cytotoxicity evaluation that proved the compounds to be non-neurotoxic at the concentrations tested, they were assayed for NRF2 induction capacity and for anti-inflammatory properties and measured by their ability to inhibit nitrite production in the lipopolysaccharide-stimulated BV2 microglial cell model. Moreover, the compounds were studied for their neuroprotective effect in a model of oxidative stress achieved by treatment of SH-SY5Y neuroblastoma cells with the rotenone–oligomycin combination and also in a model of hyperphosphorylation induced by treatment with okadaic acid. The stereocenter configuration showed a critical influence in NRF2 induction properties, and also in the neuroprotection against oxidative stress experiment, leading to the identification of the compound with S and R configuration as an interesting hit with a good neuroprotective profile against oxidative stress and hyperphosphorylation, together with a relevant anti-neuroinflammatory activity. This interesting multitarget profile will be further characterized in future work.

Eicosapentaenoic acid inhibits lipopolysaccharide (LPS)-induced nitrite production and cytokine release from J774 macrophages

Background Eicosapentaenoic acid (EPA), an omega-3 (ω-3) fatty acid, reduced cardiovascular (CV) events in high-risk patients (REDUCE-IT) but the mechanism is not fully understood. Activated macrophages, characterized by cytokine release and increased inducible nitric oxide synthase (iNOS) activity, contribute to atherosclerosis. As both a substrate for and potential inhibitor of cyclooxygenase (COX), EPA may reduce iNOS activity. Purpose The purpose of this study was to evaluate the dose-dependent effects of EPA on nitrite and cytokine release from lipopolysaccharide (LPS)-activated macrophages. Methods Murine J774 macrophages were pretreated with vehicle or EPA at 10, 20 and 40 μM for 2 h, then challenged with LPS at 1.0 μg/ml. After 24 hr, iNOS activity was measured by nitrite production using the Griess assay. EPA was compared to the COX inhibitor diclofenac at 1.0 μg/ml. Levels of interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNF-α) in cell supernatant were measured by immunochemistry using colchicine as a positive control. Results Activated macrophages caused a >4-fold increase in nitrite production (p<0.001) that was reduced by EPA in a dose-dependent manner. EPA decreased nitrite levels by 40, 62 and 77% at 10, 20 and 40 μM, respectively (p<0.01). Diclofenac separately reduced nitrite levels by 40% (p<0.01). EPA also reduced expression of IL-1β and TNF-α by 40% and 31%, respectively (p<0.01), in a manner similar to equimolar colchicine (10 μM). The reductions in IL-1β and TNF-α with EPA were dose-dependent. Conclusions EPA reduced macrophage activation as evidenced by decreased nitrite production and cytokine release similar to other anti-inflammatory agents. These findings indicate a novel effect of EPA on mechanisms of inflammation associated with vascular disease. FUNDunding Acknowledgement Type of funding sources: Private company. Main funding source(s): Amarin Pharma Inc., Elucida Research

Anti-Wrinkle Effect of Isatis indigotica Leaf Extract: Evaluation of Antioxidant, Anti-Inflammation, and Clinical Activity

Isatis indigotica leaf is an oriental herbal medicine that has been known for various pharmacological effects. However, its anti-wrinkle activity has not been fully evaluated. Therefore, we evaluated the anti-wrinkle effect of I. indigotica leaf extract on human skin. The purified extract inhibited 85.4% of 2,2-diphenyl-1-1picrylhydrazyl and 72.2% of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt radicals at a concentration of 1 mg/mL. Nitrite production was reduced by 30% after treatment with 50 μg/mL of extract. Three fractions from the extract downregulated the mRNA expression of matrix metalloproteinase-1 and -3 and upregulated the expression of interleukin 4. Among the three fractions, fraction 2 exhibited the highest activity. The major component of the extract was identified as 3,4,5-trimethoxycinnamic acid by liquid chromatography coupled with mass spectrometry. Molecular docking was conducted to predict the binding mechanism of 3,4,5-trimethoxycinnamic with matrix metalloproteinase-1 and -3, and their binding energies were −5.20 and −4.89 kcal/mol, respectively. In a clinical trial, five roughness values of visiometer and visual score were significantly reduced in treated groups compared with the placebo group after 8 weeks. I. indigotica leaf extract inhibits wrinkle formation, and could be a potential anti-wrinkle agent. This is the first clinical trial demonstrating its anti-wrinkle activity.

Differential Effects of Toll-Like Receptor Activation and Differential Mediation by MAP Kinases of Immune Responses in Microglial Cells

Microglial activation is believed to play a role in many psychiatric and neurodegenerative diseases. Based largely on evidence from other cell types, it is widely thought that MAP kinase (ERK, JNK and p38) signalling pathways contribute strongly to microglial activation following immune stimuli acting on toll-like receptor (TLR) 3 or TLR4. We report here that exposure of SimA9 mouse microglial cell line to immune mimetics stimulating TLR4 (lipopolysaccharide—LPS) or TLR7/8 (resiquimod/R848), results in marked MAP kinase activation, followed by induction of nitric oxide synthase, and various cytokines/chemokines. However, in contrast to TLR4 or TLR7/8 stimulation, very few effects of TLR3 stimulation by poly-inosine/cytidine (polyI:C) were detected. Induction of chemokines/cytokines at the mRNA level by LPS and resiquimod were, in general, only marginally affected by MAP kinase inhibition, and expression of TNF, Ccl2 and Ccl5 mRNAs, along with nitrite production, were enhanced by p38 inhibition in a stimulus-specific manner. Selective JNK inhibition enhanced Ccl2 and Ccl5 release. Many distinct responses to stimulation of TLR4 and TLR7 were observed, with JNK mediating TNF protein induction by the latter but not the former, and suppressing Ccl5 release by the former but not the latter. These data reveal complex modulation by MAP kinases of microglial responses to immune challenge, including a dampening of some responses. They demonstrate that abnormal levels of JNK or p38 signalling in microglial cells will perturb their profile of cytokine and chemokine release, potentially contributing to abnormal inflammatory patterns in CNS disease states.

Analysis of the Chemical, Antioxidant, and Anti-Inflammatory Properties of Pink Pepper (Schinus molle L.)

Here, we compared the chemical properties and antioxidant effects of black pepper (Piper nigrum L.) and pink pepper (Schinus molle L.). Additionally, the antioxidant and anti-inflammatory capacities of pink pepper were measured to determine nutraceutical potential. Pink peppers from Brazil (PPB), India (PPI), and Sri Lanka (PPS) had higher Hunter a* (redness) values and lower L* (lightness) and b* (yellowness) values than black pepper from Vietnam (BPV). Fructose and glucose were detected in PPB, PPI, and PPS, but not in BPV. PPB, PPI, and PPS had greater 2,2-diphenyl-1-picrylhydrazyl and 3-ethylbenzothiazoline-6-sulphonic acid radical scavenging stabilities and higher total phenolic contents than BPV. BPV had higher levels of piperine than the pink peppers. Gallic acid, protocatechuic acid, epicatechin, and p-coumaric acid were detected only in the three pink peppers. PPB significantly suppressed lipopolysaccharide-induced reactive oxygen species production with increased Nrf2 translocation from cytosol to nucleus and heme oxygenase-1 expression. PPB and PPS significantly suppressed lipopolysaccharide-induced nitrite production and nitric oxide synthase expression by suppressing phosphorylation of p38 without affecting cell viability. Additionally, PPB and PPS significantly suppressed ultraviolet B-induced cyclooxygenase-2 expression by affecting the phosphorylation of ERK1/2 without cell cytotoxicity. These results suggest that pink pepper is a potential nutraceutical against oxidative and inflammatory stress.

Increased Th1 Cells with Disease Resolution of Active Pulmonary Tuberculosis in Non-Atopic Patients

Type 1 CD4+ T helper (Th1) cells mediate resistance to Mycobacterium tuberculosis (Mtb), and Th2 immunity generates specific immunoglobulin E upon allergen exposure. We investigated the impact of active tuberculosis (TB), atopic status, and anti-TB treatment on the balance between Th1 and Th2 (type 2 CD4+ T helper) immunity. CD4+/interferon (IFN)-γ+ Th1 cells (%Th1) and CD4+/interleukin-4+ Th2 cells (%Th2) in bronchoalveolar lavage (BAL) fluid and peripheral blood mononuclear cells (PBMCs) were measured by flow cytometry. The BAL %Th1 was higher in TB patients at baseline, compared to that in non-TB subjects, and was further increased in TB patients after stimulation with phorbol myristate acetate and ionomycin. The stimulated BAL %Th1 was inversely correlated with the severity score of chest radiography in TB patients. Heat-killed Mtb triggered more IFN-γ and nitrite production, as determined by enzyme-linked immunosorbent assay and the Griess reaction, respectively, from the alveolar macrophages of TB patients than that of non-TB subjects. Non-atopic TB participants had a higher %Th1 in PBMCs, compared to atopic individuals, and their %Th1 decreased after 3-month anti-TB treatment. Th1 response is provoked by active TB infection, is associated with less severe radiographic changes, is reduced in atopic patients with active TB infection, and is attenuated after anti-TB treatment.