Cell IL-2 Production Research Articles

Inhibitory Effect of Eicosapentaenoic Acid on the Migration of the Esophageal Squamous Cell Carcinoma Cell Line TE-1.

Eicosapentaenoic acid (EPA) inhibits NF-ĸB activation and IL-6 production in TE-1 esophageal cancer cells. NF-ĸB is related to cancer cell migration. The aim of this study is to evaluate whether EPA has a metastasis suppressing effect. Herein, we investigated EPA-treated TE-1 cell migration using TAXIScan. EZ-TAXIScan® was used to verify whether EPA inhibits cancer cell chemotaxis. Using 50% fetal bovine serum (chemoattractant) without EPA (positive control), average velocity was 0.306±0.084 μm/min compared to 0.162±0.067 μm/min without chemoattraction (negative control). Directionalities of positive and negative controls were 1.039±0.152 and 0.488±0.251 radians, respectively, indicating a significant increase in migration of the positive control compared to that of the negative control. Average velocities were 0.306±0.084 (no EPA), 0.288±0.078 (100 μM EPA), and 0.240±0.054 200 μM (EPA) μm/min, indicating that EPA reduced velocity dose-dependently. Average directionalities were 1.039±0.152 (no EPA), 0.967±0.164 (100 μM EPA), and 0.901±0.146 (200 μM EPA) radians, indicating that EPA also inhibited directionality dose-dependently. EPA suppresses directional migration of TE-1 cells.

MiR-21a in exosomes from Lewis lung carcinoma cells accelerates tumor growth through targeting PDCD4 to enhance expansion of myeloid-derived suppressor cells.

In patients with lung cancer, myeloid-derived suppressor cells (MDSCs) have been reported to be significantly increased. Tumor-derived exosomes (TDEs) from various cancers played a critical role in MDSC induction. However, studies on the molecular mechanism underlying MDSC expansion induced by exosomes from lung cancer cells are still limited. In this study, we demonstrated that LLC-Exo accelerated tumor growth along with a significant accumulation of MDSCs in mouse tumor model. miRNA profiling showed that miR-21a was enriched in LLC-Exo. The depletion of miR-21a in LLC-Exo leads to the loss of their ability to induce MDSC expansion. Further results showed that miR-21a of LLC-Exo induced MDSC expansion via downregulation of the programmed cell death protein 4 (PDCD4) protein. The results of gain-and loss-of-function experiments validated that PDCD4 function as a critical inhibitor to negatively regulate expansion of MDSCs via inhibition Il-6 production in bone marrow cells. In addition, our data showed that exosomes derived from human lung cancer cell lines expressing miR-21a, also induced expansion of MDSCs in human CD14+ monocytes in vitro. Overall, our results demonstrated that miR-21a enriched in lung carcinoma cell-derived exosomes could promote functional expansion of MDSCs through targeting PDCD4.

Spinal cord injury can be relieved by the polysaccharides of Tricholoma matsutake by promoting axon regeneration and reducing neuroinflammation.

BackgroundWith an increase in the number of spinal cord injuries (SCIs) in China, severe dysfunction of the limb below the injured segment is prominent. Among the studies centered on the factors inducing SCIs, inflammatory response has a dramatic input on the pathogenesis of SCIs.ObjectivesThis study aimed to investigate the effects of Tricholoma matsutake polysaccharides (TMP) on function recovery following SCIs.MethodsThe cell viability, neurite growth, NF-kappa B, TNFα and IL-6 production from hydrogen peroxide-treated PC12 cells were analyzed. In-vivo, a total of 36 male C57 mice were divided into sham group, SCI group and TMP group (100 mg/kg). The protective effects of TMP were evaluated by Basso mouse scale (BMS) scores, HE staining, immunofluorescence and Western blotting.ResultsTMP promoted neurite growth and inhibited TNFα, IL-6 and NF-kappa B signaling in a concentration-dependent manner in vitro. Moreover, compared with the SCI group, the BMS scores and nerve regeneration showed a significant increase, while NF-kappa B signaling, TNFα and IL-6 production significantly decreased after TMP treatment.ConclusionTMP has a protective effect against SCIs in vitro and in vivo, which may be a potential strategy for future application in clinical practice.

Lactic acid bacterium, Lactobacillus paracasei KW3110, suppresses inflammatory stress-induced caspase-1 activation by promoting interleukin-10 production in mouse and human immune cells.

A strain of lactic acid bacteria, Lactobacillus paracasei KW3110 (KW3110), activates M2 macrophages with anti-inflammatory reactions and mitigates aging-related chronic inflammation and blue-light exposure-induced retinal inflammation in mice. However, the mechanism underlying the anti-inflammatory effects of KW3110 remains unclear. In this study, we investigated the anti-inflammatory effects of KW3110 using both mouse and human immune cells and evaluated the suppressive effect of KW3110 on the inflammatory reactions of the cells stimulated with lipopolysaccharide and adenosine 5′-triphosphate (LPS/ATP). KW3110 treatment induced anti-inflammatory cytokine interleukin (IL)-10 production in the supernatants of murine macrophage-like cells, J774A.1, and suppressed IL-1β production in the supernatants of LPS/ATP-stimulated cells. The influence of KW3110 on the production of these cytokines was inhibited by pre-treatment with phagocytosis blocker or transfection with siRNAs for IL-10 signaling components. KW3110 treatment also suppressed activation of caspase-1, an active component of inflammasome complexes, in LPS/ATP-stimulated J774A.1 cells, and its effect was inhibited by transfection with siRNAs for IL-10 signaling components. In addition to the effects of KW3110 on J774A.1 cells, KW3110 treatment induced IL-10 production in the supernatants of human monocytes, and KW3110 or IL-10 treatment suppressed caspase-1 activation and IL-1β production in the supernatants of LPS/ATP-stimulated cells. These results suggest that KW3110 suppresses LPS/ATP stimulation-induced caspase-1 activation and IL-1β production by promoting IL-10 production in mouse and human immune cells. Our findings reveal a novel anti-inflammatory mechanism of LAB and the effect of KW3110 on caspase-1 activation is expected to contribute to constructing future preventive strategies for inflammation-related disorders using food ingredients.

Abstract 4513: The role of HPK1 in the regulation of T cell function and anti-tumor immune activity

Abstract Antigen recognition and T-cell receptor (TCR) activation are fundamental processes that drive anti-tumor T cell responses. HPK1 has been identified as a negative regulator of TCR activation, as well as BCR activation, and is a potential anticancer target for immuno-oncology. We show that genetic ablation of HPK1 in human T cells resulted in increased cytokine production upon cell stimulation. Jurkat cells with genetic HPK1 knockout were unable to phosphorylate SLP76, a direct downstream target of HPK1, upon cell stimulation which was associated with increased IL-2 production. Genetic deletion of HPK1 in human RAMOS B cells reduced phosphorylation of BLNK upon IgM stimulation and led to increased TNF alpha and TNF beta production. Treatment of HPK1 knockout primary human T cells with pembrolizumab enhanced IFN gamma secretion compared to the knockout cells alone. Based on these genetic data, HPK1 may be an attractive target for immuno-oncology. We describe herein the in vitro and in vivo profile of several small molecule HPK1 inhibitors and report a surprising disparity between in vitro and in vivo findings. In vitro, the HPK1 inhibitors tested phenocopied the genetic data by potently inhibiting pSLP76 and enhancing IL-2 production in Jurkat cells following stimulation. Similarly, HPK1 inhibitors enhanced IL-2 production in human PBMCs, and increased IFN gamma production in combination with atezolizumab in a primary T cell co-culture assay. In vivo, the HPK1 small molecule inhibitors inhibited tumor growth in the MC38 model, which was further enhanced in combination with anti-PD-L1 in 3 different syngeneic models (MC38, CT26 and MBT-2). Despite these results, tumor growth inhibition was not observed in the GL261 glioma syngeneic model whose growth was recently shown to be inhibited when implanted into HPK1 kinase-dead mice. Further, although several in vitro assays demonstrated increased functional cytokine production with HPK1 inhibitor treatment, the compounds did not increase in vivo cytokine production in tumors. Moreover, combining HPK1 inhibitors with a PD-L1 antibody in vivo abolished the anti-PD-L1-induced production of IFN gamma in the CT26 model. In summary, while our genetic data support the role of HPK1 as a negative regulator of T and B cells, the in vitro activity of HPK1 kinase activity inhibitors was not correlated with functional effects in in vivo syngeneic tumor models. These results underline the complexity of interpreting HPK1 biology and also highlight challenges for the development of clinically active compounds targeting this pathway. Citation Format: Yaoyu Chen, Jonathan Rios-Doria, Michelle Pusey, Kerri Lasky, Min Ye, Pramod Thekkat, Karen Gallagher, Kristine Stump, Patricia Conlen, Christine Gardiner, Hui Wang, Alexander Sokolsky, Mark Rupar, Luping Lin, Elham Behshad, Maryanne Covington, Holly Koblish, Oleg Vechorkin, Wenqing Yao, Sunkyu Kim, Yingnan Chen. The role of HPK1 in the regulation of T cell function and anti-tumor immune activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4513.

Abstract 1550: Single-cell RNA-Seq identified an immunosuppressive IL-17 producing tumor-infiltrating T cells in murine gastric tumor model

Abstract BACKGROUND: YTN2 and YTN16 cells are chemically induced gastric cancer cell lines. YTN2 is spontaneously rejected in C57BL/6 mice in CD8+ T cell-dependent manner, while YTN16 grows progressively. To investigate the interaction of tumor microenvironment and T cells, bulk RNA-Seq of total tumor RNA and single-cell RNA-Seq of tumor-infiltrating T cells were performed in YTN2 and YTN16 gastric tumor-bearing mice. METHOD: Tumor cells (5×106) were subcutaneously inoculated into C57BL/6 mice. On day 7, tumors were harvested, and total tumor RNA and tumor-infiltrating lymphocytes were isolated. CD8+ and CD4+ cells were sorted from YTN2 and YTN16 tumor tissues. Single-cell RNA-sequencing analysis was performed using vertical flow array chips (VAFC) for 44 T cell-related genes. IL-17 production in tumor-infiltrating T cells was evaluated by intracellular cytokine staining. Anti-IL-17(clone 17F3) and anti-PD-1(RMP1-14) mAbs were injected into YTN16 tumor-bearing mice on days 3, 6, 9, and 12 and days 6, 9, and 12, respectively. RESULT: Bulk RNA-Seq revealed that higher gene expression of immune-related genes, including cytokines, chemokines, their receptors and checkpoint molecules, were detected in YTN2 tumors than YTN16. IL-17 expression was not detected in YTN2 or YTN16. We analyzed 1142 tumor infiltrating CD4+ and CD8+ T cells isolated from YTN2 and YTN16 tumor tissues. Unsupervised clustering of all T cells using the Louvain method identified 7 clusters, including 3 clusters (clusters 1, 4, 5) for CD8+ and 3 clusters (clusters 2, 3, 6, 7) for CD4+ T cells. Clusters 1-6 included T cells from both YTN2 and YTN16. Cluster 7 consisted of CD4+ T cells only from YTN16. CD8+ T cells in cluster 5 highly expressed Slc2a1, Ifng, Tnf, Fasl, Cxcr3, Il2rb, Il2rg, Il7r, Pdcd1, Tigit, Eomes, Bcl2, and Tbx21. In contrast, these genes were scarcely expressed in cluster 4. Cluster 1 was an intermediate phenotype of these two clusters. CD8+ T cells from YTN16, but not YTN2 in cluster 5, expressed Il17. CD4+ T cells in cluster 2 expressed Slc2a1, Tnfrsf9, Pdcd1, Clta4, Tigit, Hif-1a and Icos at high level. Cluster 2 CD4+ T cells from YTN2 expressed more Havcr2 and Tbx21 and less Il17 than those from YTN16. T cells in cluster 3 did not express these genes. Cluster 7 was characterized by high Il17 expression. By single-cell RNA-Seq analysis, IL-17-producing cells were only detected in YTN16 tumors. Consistent with single-cell analysis, we detected IL-17 producing cells in the YTN16 tumor by flow cytometry. To elucidate the function of IL-17 in the tumor, we used anti-IL-17 blocking mAb and showed that blocking of IL-17 suppressed YTN16 tumor growth. Combination therapy of anti-IL-17 and anti-PD-1 enhanced the anti-tumor effect. CONCLUSION: Single-cell RNA-sequencing analysis, but not bulk RNA-Seq, identified IL-17-producing CD4+ T cells in YTN16 tumors and revealed an immunosuppressive role of IL-17 in progressive cancer. Citation Format: Masataka Shirai, Koji Nagaoka, Kiyomi Taniguchi, Changbo Sun, Akihiro Hosoi, Kazuhiro Kakimi. Single-cell RNA-Seq identified an immunosuppressive IL-17 producing tumor-infiltrating T cells in murine gastric tumor model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1550.

Zbtb7b suppresses aseptic inflammation by regulating m6A modification of IL6 mRNA

Radiotherapy is a crucial approach for treating tumors. However, radiation-induced aseptic inflammation is a common complication. Radiation pneumonitis is the acute manifestation of radiation-induced lung disease, and interleukin 6 (IL-6) is a major proinflammatory cytokine involved in radiation-induced lung injury. Here we found that silencing Zinc finger and BTB domain-containing protein 7B (Zbtb7b) resulted in higher radiation-induced IL-6 production in THP1 cells and BEAS-2B lung bronchial epithelial cells. Mechanistically, Zbtb7b recruited RNA demethylase ALKBH5 to IL6 mRNA. Subsequentially, it demethylated N6-methyladenosine (m6A) modification of IL6 mRNA and inhibited its nuclear export. Thus, Zbtb2b epigenetically suppresses irradiation-induced IL-6 production in the lungs via inhibiting the m6A modification and nucleocytoplasmic transport of IL6 mRNA, serving as a new potential predictive marker and therapeutic target in radiation pneumonitis treatment.

Cannabidiol (CBD): a killer for inflammatory rheumatoid arthritis synovial fibroblasts

Cannabidiol (CBD) is a non-intoxicating phytocannabinoid from cannabis sativa that has demonstrated anti-inflammatory effects in several inflammatory conditions including arthritis. However, CBD binds to several receptors and enzymes and, therefore, its mode of action remains elusive. In this study, we show that CBD increases intracellular calcium levels, reduces cell viability and IL-6/IL-8/MMP-3 production of rheumatoid arthritis synovial fibroblasts (RASF). These effects were pronounced under inflammatory conditions by activating transient receptor potential ankyrin (TRPA1), and by opening of the mitochondrial permeability transition pore. Changes in intracellular calcium and cell viability were determined by using the fluorescent dyes Cal-520/PoPo3 together with cell titer blue and the luminescent dye RealTime-glo. Cell-based impedance measurements were conducted with the XCELLigence system and TRPA1 protein was detected by flow cytometry. Cytokine production was evaluated by ELISA. CBD reduced cell viability, proliferation, and IL-6/IL-8 production of RASF. Moreover, CBD increased intracellular calcium and uptake of the cationic viability dye PoPo3 in RASF, which was enhanced by pre-treatment with TNF. Concomitant incubation of CBD with the TRPA1 antagonist A967079 but not the TRPV1 antagonist capsazepine reduced the effects of CBD on calcium and PoPo3 uptake. In addition, an inhibitor of the mitochondrial permeability transition pore, cyclosporin A, also blocked the effects of CBD on cell viability and IL-8 production. PoPo3 uptake was inhibited by the voltage-dependent anion-selective channel inhibitor DIDS and Decynium-22, an inhibitor for all organic cation transporter isoforms. CBD increases intracellular calcium levels, reduces cell viability, and IL-6/IL-8/MMP-3 production of RASF by activating TRPA1 and mitochondrial targets. This effect was enhanced by pre-treatment with TNF suggesting that CBD preferentially targets activated, pro-inflammatory RASF. Thus, CBD possesses anti-arthritic activity and might ameliorate arthritis via targeting synovial fibroblasts under inflammatory conditions.

Association between opioid use disorder (tramadol) and serum level of interleukin-2

BackgroundTramadol hydrochloride is a centrally acting analgesic drug that has turned out to be a drug of growing interest due to its success in the management of pain in humans. By considering the twin mechanism of action of tramadol, as the activation of the opioid and of the monoaminergic systems, the potential immunological effects of this drug should be considered and be evaluated. Studies show that Tramadol induces an improvement of postoperative immune suppression and increases NK cell activity, lymphocyte proliferation, and IL-2 production which may be linked to its addictive potentiality. The present study aims to investigate the possible association between opioid use disorder (tramadol) and the serum level of interleukin-2 (IL-2).An observational cross sectional study conducted at the Outpatient Clinic and Inpatient Unit of the Addiction Management Unit of the Neurology and Psychiatry Department of Assiut University Hospital. Assessment of serum level of IL-2 for 59 male patients diagnosed with opioid use disorder (tramadol) according to DSM-5 compared to 32 cross-matched male individuals as a control group.ResultsComparing the serum level of IL-2 in patients with opioid use disorder (tramadol) with that of the controls, it was found that the patients with opioid use disorder (tramadol) have significantly higher serum level of IL-2 (318.69 ± 35.89) than that of controls (260.45 ± 30.08) (P = 0.01). Patients with tramadol urine assay (> 200 ng/ml) have significantly higher serum level of IL-2.ConclusionThe current study found that patients with opioid use disorder (tramadol) have significantly elevated serum levels of IL-2 than healthy controls. Also, this elevation was dose related.

Blocking VEGF signaling augments interleukin-8 secretion via MEK/ERK/1/2 axis in human retinal pigment epithelial cells.

To identify proangiogenic factors engaged in neovascular age-related macular degeneration (AMD) except vascular endothelial growth factor (VEGF) from human retinal pigment epithelial (hRPE) cells and investigate the underlying mechanisms. VEGF receptor 2 (VEGFR2) in ARPE-19 cells was depleted by siRNA transfection or overexpressed through adenovirus infection. The mRNA and the protein levels of interleukin-8 (IL-8) in ARPE-19 cells were measured by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay respectively. The protein levels of AKT, p-AKT, MEK, p-MEK, ERK1/2, p-ERK1/2, JNK, p-JNK, p38 and p-p38 were detected by Western blotting. A selective chemical inhibitor, LY3214996, was employed to inhibit phosphorylation of ERK1/2. Cell viability was determined by MTT assay. Knockdown of VEGFR2 in ARPE-19 cells robustly augmented IL-8 production at both the mRNA and the protein levels. Silencing VEGFR2 substantially enhanced phosphorylation of MEK and ERK1/2 while exerted no effects on phosphorylation of AKT, JNK and p38. Inhibiting ERK1/2 phosphorylation by LY3214996 reversed changes in VEGFR2 knockdown-induced IL-8 upregulation at the mRNA and the protein levels with no effects on cell viability. VEGFR2 overexpression significantly reduced IL-8 generation at the mRNA and the protein levels. Blockade of VEGF signaling augments IL-8 secretion via MEK/ERK1/2 axis and overactivation of VEGF pathway decreases IL-8 production in hRPE cells. Upregulated IL-8 expression after VEGF signaling inhibition in hRPE cells may be responsible for being incompletely responsive to anti-VEGF remedy in neovascular AMD, and IL-8 may serve as an alternative therapeutic target for neovascular AMD.

Early modulation of macrophage ROS-PPAR\u03b3-NF-\u03baB signalling by sonodynamic therapy attenuates neointimal hyperplasia in rabbits

Disruption of re-endothelialization and haemodynamic balance remains a critical side effect of drug-eluting stents (DES) for preventing intimal hyperplasia. Previously, we found that 5-aminolevulinic acid-mediated sonodynamic therapy (ALA-SDT) suppressed macrophage-mediated inflammation in atherosclerotic plaques. However, the effects on intimal hyperplasia and re-endothelialization remain unknown. In this study, 56 rabbits were randomly assigned to control, ultrasound, ALA and ALA-SDT groups, and each group was divided into two subgroups (n = 7) on day 3 after right femoral artery balloon denudation combined with a hypercholesterolemic diet. Histopathological analysis revealed that ALA-SDT enhanced macrophage apoptosis and ameliorated inflammation from day 1. ALA-SDT inhibited neointima formation without affecting re-endothelialization, increased blood perfusion, decreased the content of macrophages, proliferating smooth muscle cells (SMCs) and collagen but increased elastin by day 28. In vitro, ALA-SDT induced macrophage apoptosis and reduced TNF-α, IL-6 and IL-1β via the ROS-PPARγ-NF-κB signalling pathway, which indirectly inhibited human umbilical artery smooth muscle cell (HUASMC) proliferation, migration and IL-6 production. ALA-SDT effectively inhibits intimal hyperplasia without affecting re-endothelialization. Hence, its clinical application combined with bare-metal stent (BMS) implantation presents a potential strategy to decrease bleeding risk caused by prolonged dual-antiplatelet regimen after DES deployment.

IL-10-producing NK cells exacerbate sublethal Streptococcus pneumoniae infection in the lung

Lung inflammation is tightly controlled to balance microbial clearance with the tissue damage that accompanies this response. Bacterial pathogens including Streptococcus pneumoniae (S. pneumoniae) modulate immune regulation by promoting secretion of the anti-inflammatory cytokine IL-10. The important cellular sources of IL-10 that impact protection against different bacterial infections are not well characterized. We find that S. pneumoniaeactivates IL-10 secretion from natural killer (NK) cells in the lung, which restrict host protection in a mouse model of sublethal infection. Direct transfer of wild-type NK cells into the lungs of IL-10-deficient mice drives bacterial expansion, identifying NK cells as a critical source of IL-10 promoting S. pneumoniae infection. The S. pneumoniae virulence protein Spr1875 was found to elicit NK cell IL-10 production in purified cells and in the lungs of live animals. These findings reveal therapeutic targets to combat bacterial-driven immune regulation in the lung.

Inhibitory Effect of Lipoteichoic Acid Derived from Three Lactobacilli on Flagellin-Induced IL-8 Production in Porcine Peripheral Blood Mononuclear Cells.

Probiotics in livestock feed supplements are considered to be an alternative to antibiotics. However, effector molecules responsible for the beneficial roles of probiotics in pigs are in general not well known. Thus, this study demonstrated that a well-known virulence factor, flagellin of Salmonella typhimurium, significantly induced IL-8 production in porcine peripheral blood mononuclear cells, whereas lipoteichoic acid (LTA), a major cell wall component of Gram-positive bacteria Lactobacillus plantarum, L. casei, and L. rhamnosus GG, effectively inhibited flagellin-induced IL-8 production at mRNA and protein levels. However, the lipoproteins of L. plantarum, L. casei, and L. rhamnosus GG did not suppress flagellin-induced IL-8 production. While D-alanine-deficient L. plantarum LTA inhibited flagellin-induced IL-8 production, L. plantarum LTA deficient in both D-alanine and acyl chains failed to inhibit it; this suggests that the acyl moieties of L. plantarum LTA are essential for inhibiting flagellin-induced IL-8 production. Taken together, L. plantarum LTA plays an important role in improving anti-inflammatory responses of porcine peripheral blood mononuclear cells.

Curcumin attenuates IL-17A mediated pulmonary SMAD dependent and non-dependent mechanism during acute lung injury in vivo.

Acute lung injury (ALI) is a pathologic condition responsible for incurable human chronic respiratory diseases. Recent studies have shown the involvement of the glycoprotein, IL17A secreted by IL-17 producing cells in chronic inflammation. The current investigation was carried out to study the IL-17A mediated activation of SMAD and non- SMAD signaling in alveolar epithelial cells and to assess the putative modulatory role of curcumin. C57BL/6 mice were exposed to IL-17A and curcumin was administered as an intervention to modulate the IL-17A-induced alveolar damage. Techniques like Immunofluorescence and real-time PCR were used. We found elevated expression of IL-17A and IL-17A-associated signaling pathways to be activated in mice lung tissues. Curcumin intervention in vivo promoted the resolution of IL-17A-induced ALI and attenuated pulmonary damage. Increase phosphorylation of non- SMAD proteins like P-EGFR, P-STAT-1, STAT-3, P-JAK-1/2, P-JNK, and also SMAD proteins like P- SMAD 2/3 and TGF-β1 was encountered upon IL-17A exposure, while curcumin intervention reversed the protein expression levels. Curcumin was found to block mRNA expressions of non- SMAD genes EGFR, JNK-1, JAK1, JAK2, STAT-1, STAT-3, MAPK14, also of TGF-β1 and SMAD genes like SMAD 2, SMAD 3. However, mRNA expressions of SMAD 6 and SMAD 7 were increased upon curcumin intervention. Our study indicates that IL-17A participates in the development of ALI in both SMAD dependent and independent manner and the IL-17A signaling components were effectively controlled by curcumin, suggesting probable anti-inflammatory use of curcumin during ALI.

P0523PROTECTIVE ROLE OF REGULATORY B CELLS ON THE RENAL TISSUE REPAIRMEN AFTER ISCHEMIA REPERCUSSION INJURY

Abstract Background and Aims Acute kidney injury (AKI), even if followed by renal recovery, is a risk factor for the future development of chronic kidney disease (CKD) and end- stage renal disease. It has been postulated that interleukin-10 (IL-10)-producing Regulatory B cells (Breg) play an important role for the tissue repairment in several tissues and organs. Basically, protective role of Breg has been reported in inflammatory bowel disease. In the kidney, it has been shown that IL-10 suppresses renal function decline and improves renal prognosis in IRI model, a typical model of AKI. However, the identity of Breg in the kidney and their origin have not been clarified. Further, how the Breg works during the transition from AKI to CKD is not known. Therefore, first we investigated whether Breg existed in renal tissue on the progression from AKI to CKD in IRI model mice. Further, we performed splenectomy, and examined the renal injury, Breg, and plasma IL-10 levels in this model. Method To examine the existence of Breg in the kidney of IRI model, we used 8-10 weeks-old GFP / IL-10 mice based on C57BL / 6J mice. They are reporter mice for IL-10 producing cells, and can visualize IL-10 producing cells under a fluorescence microscope without fluorescent immunostaining. We prepared following three groups, sham, IRI (unilateral), and IRI + SN (splenectomy) groups. Mice were anesthetized with chloral hydrate (4 g/kg,, intraperitoneal). After making a midline incision, exposed a blood vessel of the left renal pedicles and clamped it for 30 min by clips. one day, 7 days, and 14 days after the surgery, mice were sacrificed, and renal function and plasma IL-10 levels as well as tissue damages by PAS and Masson’s Trichrome staining were assessed. Tissue IL-10-producing cells were detected by flow cytometry. Results There was no difference of plasma IL-10 levels and renal tubulointerstitial injury in IRI group and IRI+SN group on day 1 after IRI. However, on day 7 and day 14, plasma IL-10 levels became gradually higher in IRI group, and SN decreased the increase in IL-10 levels. Tubulointerstitial injury was induced by IRI and SN further worsened tubular damages. Serum Cr and BUN levels were not different in three groups due to normal right kidney. On day 1, number of IL-10-producing B cells increased in the spleen and renal medulla in IRI group confirmed by flow cytometry, which was completely diminished by SN, suggesting that origin of the infiltrated Breg might be spleen, thereby being involved in the protective role in IRI injury in the kidney. Conclusion We report for the first time that Breg might be recruited from spleen by AKI, which may be one of the mechanisms to prevent the progression to CKD.

Effect of Supplementations with Saccharomyces cerevisiae and β-Glucans in Pregnant Recipient Mares on the Colostrum quality: Preliminary Results

Pythiosis is a life-threatening disease caused by the fungus-like microorganism Pythium insidiosum that can lead to death if not treated. Since P. insidiosum has particular cell wall characteristics, pythiosis is difficult to treat, as it does not respond well to traditional antifungal drugs. In our study, we investigated a new immunotherapeutic approach with potential use in treatment and in the acquisition of immunity against pythiosis. Dendritic cells from both human and mouse, pulsed with P. insidiosum heat-inactivated zoospore, (1,3)(1,6)-β-glucan and the immunotherapeutic PitiumVac® efficiently induced naïve T cell differentiation in a Th1 phenotype by the activation of specific Th1 cytokine production in vitro. Heat-inactivated zoospores showed the greatest Th1 response among the tested groups, with a significant increase in IL-6 and IFN-γ production in human cells. In mice cells, we also observed a Th17 pathway induction, with an increase on the IL-17A levels in lymphocytes cultured with β-glucan pulsed DCs. These results suggest a potential use of DCs pulsed with P. insidiosum antigens as a new therapeutic strategy in the treatment and acquisition of immunity against pythiosis.

Mechanism of azithromycin in airway diseases.

Azithromycin (AZM) has been used to treat chronic inflammatory airway diseases because it regulates cell–cell contact between airway epithelial cells. Airway mucus hypersecretion is an important component of chronic respiratory diseases. Mucin 5AC (MUC5AC) is the major mucin produced by airway epithelial cells, and hypersecretion of MUC5AC is a sign of various pulmonary inflammatory diseases. Recently, it was found that matrix metallopeptidase 9 is involved in mucus hypersecretion. Moreover, AZM can inhibit the ability of TNF-α-to induce interleukin (IL)-8 production. This review focuses on the effects on AZM that may be beneficial in inhibiting MUC5AC, matrix metalloprotease-9 and IL-8 production in airway epithelial cells. In addition, recent studies have begun to assess activation of mitogen-activated protein kinase (MAPK) signaling pathways in response to AZM. Understanding these new developments may be helpful for clinicians.

6,6′-Aryl trehalose analogs as potential Mincle ligands

6,6′-Aryl trehalose derivatives have been synthesized with a view towards identifying novel Th-17-inducing vaccine adjuvants based on the high affinity Mincle ligand Brartemicin. The initial structure-activity relationships of these novel trehalose-based compounds were investigated. All compounds have been evaluated for their ability to engage the Mincle receptor and induce a potential pro-Th17 cytokine profile from human peripheral blood mononuclear cells based on IL-6 production in human peripheral blood mononuclear cells. The preliminary biological characterization of the designed analogs presented in this paper should aid in the future design and testing of more affine ligands that may foster the discovery of novel adjuvants with improved pharmacological properties.

A Unique Enzymatically Hydrolyzed Whey Protein Positively Impacts Measures of Immunity

Abstract Objectives Milk proteins appear to have little direct impact on the immune system when consumed directly. The digestive system breaks them down into absorbable size and they provide nutritive value. However, it has been discovered that enzymatically hydrolyzed peptides can more directly influence the immune system, presumably before digestion completes their hydrolysis during gut immune system exposure. The term ‘bioactive centers’ will be used to describe these unique peptides. Methods After screening several different versions of enzyme hydrolyzed whey, a new bioactive center-containing product was found that had promise for further immunological research. It was compared against transfer factors, 4Life Research's colostrum filtrate, which have previously been studied for their immune activity. Results In a natural killer cell activity assay, the bioactive centers product was significantly (70%) more effective than IL-2, the standard stimulant, and equally as effective as transfer factors. A second experiment confirmed that this effect was consistent across different production batches of the product. A third experiment found that a combination of the bioactive centers and transfer factors enhanced the activity by 10% on average. In a second study, the bioactive centers were evaluated in a mouse model. They were as effective as transfer factors in stimulating NK cell activity, antibody production, TNF-alpha and IL-2 production, and in stimulating phagocytosis. In most cases, results were higher, though not statistically significantly so, for the transfer factor/bioactive centers combination. Conclusions Since the discovery of transfer factors, work both in and outside of 4Life Research has continued to try to identify new approaches to positively impacting the immune system. These results show that bioactive centers from whey can have such activity. Further work is needed to fully understand their impact in human health. Funding Sources 4Life Research.

B cell-derived IL-1β and IL-6 drive T cell reconstitution following lymphoablation.

Understanding the mechanisms of T cell homeostatic expansion is crucial for clinical applications of lymphoablative therapies. We previously established that T cell recovery in mouse heart allograft recipients treated with anti-thymocyte globulin (mATG) critically depends on B cells and is mediated by B cell-derived soluble factors. B cell production of interleukin (IL)-1β and IL-6 is markedly upregulated after heart allotransplantation and lymphoablation. Neutralizing IL-1β or IL-6 with mAb or the use of recipients lacking mature IL-1β, IL-6, IL-1R, MyD88, or IL-6R impair CD4+ and CD8+ T cell recovery and significantly enhance the graft-prolonging efficacy of lymphoablation. Adoptive co-transfer experiments demonstrate a direct effect of IL-6 but not IL-1β on T lymphocytes. Furthermore, B cells incapable of IL-1β or IL-6 production have diminished capacity to mediate T cell reconstitution and initiate heart allograft rejection upon adoptive transfer into mATG treated B cell deficient recipients. These findings reveal the essential role of B cell-derived IL-1β and IL-6 during homeostatic T cell expansion in a clinically relevant model of lymphoablation.