Immune Cell Cytokine Production Research Articles

Bioinformatics analyses of infiltrating immune cell participation on pancreatic ductal adenocarcinoma progression and in vivo experiment of the therapeutic effect of Shuangshen granules

Ethnopharmacological relevanceShuangshen granules (SSG), a nationally patented Chinese medicinal formula, including Panax quinquefolium L., Panax notoginseng (Burkill) F. H. Chen, and Cordyceps sinensis (Berk.) Sacc., has demonstrated remarkable therapeutic effects on pancreatic cancer in clinical treatment for nearly 10 years. Previous pharmacological researches have found that its main components, including ginsenosides and cordycepin have anticancer or preventive effects on pancreatic ductal adenocarcinoma (PDAC), which may be associated with immune metabolism. However, the underlying pharmacological mechanism of SSG in the truncation effect of PDAC progression is still unclear. Aim of the studyTo comprehensively understand the infiltrating immune cells during the different stages of the PDAC development chain and search for immune-related biomarkers that could potentially serve as drug targets through bioinformatic analysis. Meanwhile, the truncation effect of SSG on PDAC progression was also investigated. Materials and methodsThe gene expression profiles at different PDAC developmental stages, including normal pancreas, pancreatic intraepithelial neoplasia (PanIN), and PDAC, were retrieved from the GEO database. The GEO2R tool was used to identify differentially expressed genes among the three groups. Functional enrichment analysis was performed with the GSEA software and Metascape platform. The CIBERSORT algorithm evaluated immune cell infiltration in the three groups, and immune-related biomarkers were identified. Correlation analysis was employed to examine the association between immune cells and the biomarkers. One of these biomarkers was selected for immunohistochemistry validation in human samples. Lastly, the effectiveness of SSG against PDAC progression and the influence on the selected biomarker were validated in vivo. The underlying pharmacological mechanisms were also explored. ResultsOne dataset was obtained, where the functional enrichment of DEGs primarily involved immune effector processes and cytokine production of immune cells. The differential immune cells reflected during the progression from PanIN to PDAC were B memory cells, monocytes, M2 macrophages, and activated dendritic cells. The upregulation of ACTA2 was closely associated with M2 macrophage regulation. The immunohistochemistry on human samples validated significant differences in ACTA2 expression levels as the PDAC progressed. Moreover, animal experiments revealed that the national patented drug SSG ameliorated the pathological changes, decreased the expression of ACTA2 and its functional protein α-smooth muscle actin during PDAC progression. The underlying pharmacological mechanism was related to the regulation of macrophage polarization and downregulation of TGF-β/Smad signaling pathway. ConclusionsThe immunosuppressive environment changes during the PDAC progression. ACTA2 is a potential immuned-target for drug prevention of PDAC, while SSG could be a promising drug candidate.

1724-P: Caspase 3/7 Activation and RIPK3 Are Dispensable for Synthetic hIAPP-Induced ß-Cell Death

Islet amyloid is a hallmark of type 2 diabetes that is associated with β-cell loss. Aggregation of the β-cell secretory product human islet amyloid polypeptide (hIAPP) to form islet amyloid promotes β-cell death 1) via direct effects on the β cell and 2) by stimulating islet immune cell cytokine production. We recently showed that β cells are susceptible to TNFα-induced necroptosis, a form of caspase-independent, receptor interacting protein kinase 3 (RIPK3)-mediated cell death. We hypothesized that RIPK3 also promotes β-cell death in response to islet amyloid formation. To test this hypothesis, we evaluated INS-1, NIT-1 and isolated mouse islet cells treated with synthetic hIAPP (20 μM) +/− the pan-caspase inhibitor zVAD (40 μM). We found synthetic hIAPP increased cell death and caspase activity in INS-1, NIT-1 and mouse islet cells over 48 h. Remarkably, inhibition of caspases with zVAD (as measured by caspase 3/7 activity) failed to reduce synthetic hIAPP-mediated cell death in INS-1 (n=3, p=0.99), NIT-1 (n=3, p=0.99) or mouse islet cells (n=4, p=0.98). To evaluate RIPK3 in hIAPP-induced β-cell death, we examined a small molecule RIPK3 inhibitor (GSK’872, 5 μM), gene-edited NIT-1 Ripk3Δ cells (gRNA targeting exon 6), and intact isolated Ripk3−/− mouse islets. We found GSK’872 failed to protect INS-1 (n=3, p=0.32) or NIT-1 (n=3, p=0.30) cells from hIAPP-induced cell death over 48 h. Similarly, neither NIT-1 Ripk3Δ cells (n=2, hIAPP: p=0.82, hIAPP+zVAD: p=0.97) nor Ripk3−/− islets (n=4, hIAPP: p=0.63, hIAPP+zVAD: p=0.86) were protected from synthetic hIAPP-induced cell death either when caspases were active or inhibited. Our data indicate that caspase 3/7 activity and RIPK3 are dispensable for synthetic hIAPP-induced β-cell death in vitro. However, we found that RIPK3 is upregulated in response to endogenous islet amyloid formation in vitro. Future studies will determine whether RIPK3 promotes β-cell loss in part via effects on endogenous amyloid-induced cytokine signaling. Disclosure N.Mukherjee: None. C.J.Contreras: None. L.Lin: None. E.P.Cai: None. S.E.Kahn: Advisory Panel; Anji Pharmaceuticals, Bayer Inc., Boehringer Ingelheim Inc., Eli Lilly and Company, Merck & Co., Inc., Other Relationship; Novo Nordisk. A.T.Templin: None. Funding U.S. Department of Veterans Affairs (IK2BX004659 to A.T.T.); National Institutes of Health (T32DK064466 to C.J.C.); Indiana University School of Medicine (to N.M.)

NLRP3 inhibitor dopamine receptors agonist as a potential therapeutic strategy for treatment of overactive immune responses in COVID-19

Patients infected with coronavirus disease 2019 (COVID-19) have high serum levels of proinflammatory cytokines. The “cytokine storm” has become one of the major causes of death for critically ill patients infected by COVID-19. Glucocorticoids, plasma from convalescent patients, blood purification, and tocilizumab are currently recommended for use when the body’s inflammatory response is overactivated. However, there are limitations in terms of medicinal effects, equipment reserves, and treatment expense. These challenges prompted us to assess classical agents with good safety and mature production technology. A recent study showed that nucleotide-binding oligomerization domain (NOD)-, leucine-rich repeat (LRR)-, and pyrin domain-containing protein 3 (NLRP3) inflammasomes drive COVID-19 pathology. We speculate that suppression of NLRP3 inflammasome-derived cytokine production may be beneficial in COVID-19–infected patients. Dopamine receptors are present in almost all immune cells and can modulate their activation, proliferation, and cytokine production of immune cells. Previous studies have shown that dopamine receptor agonists can control systemic inflammation through inhibition of the NLRP3 inflammasome. This suggests that dopamine receptor agonists may be a new strategy for the treatment of overactive immune responses in COVID-19 patients. This is worthy of further investigation in clinical practice.

The NLRP3 inflammasome contributes to inflammation-induced morphological and metabolic alterations in skeletal muscle.

Systemic inflammation is associated with skeletal muscle atrophy and metabolic dysfunction. Although the nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome contributes to cytokine production in immune cells, its role in skeletal muscle is poorly understood. Here, we studied the link between inflammation, NLRP3, muscle morphology, and metabolism in in vitro cultured C2C12 myotubes, independent of immune cell involvement. Differentiated C2C12 myotubes were treated with lipopolysaccharide (LPS; 0, 10, and 100-200ng/mL) to induce activation of the NLRP3 inflammasome with and without MCC950, a pharmacological inhibitor of NLRP3-induced IL-1β production. We assessed markers of the NLRP3 inflammasome, cell diameter, reactive oxygen species, and mitochondrial function. NLRP3 gene expression and protein concentrations increased in a time-dependent and dose-dependent manner. Intracellular IL-1β concentration significantly increased (P<0.0001), but significantly less with MCC950 (P=0.03), suggestive of moderate activation of the NLRP3 inflammasome in cultured myotubes upon LPS stimulation. LPS suppressed myotube growth after 24h (P=0.03), and myotubes remained smaller up to 72h (P=0.0009). Exposure of myotubes to IL-1β caused similar alterations in cell morphology, andMCC950 mitigated these LPS-induced differences in cell diameter. NLRP3 appeared to co-localize with mitochondria, more so upon exposure to LPS. Mitochondrial reactive oxygen species were higher after LPS (P=0.03), but not after addition of MCC950. Myotubes had higher glycolytic rates, and mitochondria were more fragmented upon LPS exposure, which was not altered by MCC950 supplementation. LPS-induced activation of the NLRP3 inflammasome in cultured myotubes contributes to morphological and metabolic alterations, likely due to its mitochondrial association.

High-CBD Extract (CBD-X) Downregulates Cytokine Storm Systemically and Locally in Inflamed Lungs.

Cytokine storm refers to the dysregulated production of inflammatory mediators leading to hyperinflammation. They are often detrimental, and worsen the severity of COVID-19 and other infectious or inflammatory diseases. Cannabinoids are known to have anti-inflammatory effects but their possible therapeutic value on cytokine storms has not been fully elucidated. In vivo and ex vivo studies were carried out to investigate the effects of high-THC and high-CBD extracts on cytokine production in immune cells. Significant differences between the extracts were observed. Subsequent experiments focusing on a specific high CBD extract (CBD-X) showed significant reductions in pro-inflammatory cytokines in human-derived PBMCs, neutrophils and T cells. In vivo mouse studies, using a systemically inflamed mouse model, showed reductions in pro-inflammatory cytokines TNFα and IL-1β and a concurrent increase in the anti-inflammatory cytokine IL-10 in response to CBD-X extract treatment. Lung inflammation, as in severe COVID-19 disease, is characterized by increased T-cell homing to the lungs. Our investigation revealed that CBD-X extract impaired T-cell migration induced by the chemoattractant SDF1. In addition, the phosphorylation levels of T cell receptor (TCR) signaling proteins Lck and Zap70 were significantly reduced, demonstrating an inhibitory effect on the early events downstream to TCR activation. In a lung inflamed mouse model, we observed a reduction in leukocytes including neutrophil migration to the lungs and decreased levels of IL-1β, MCP-1, IL-6 and TNFα, in response to the administration of the high-CBD extract. The results presented in this work offer that certain high-CBD extract has a high potential in the management of pathological conditions, in which the secretion of cytokines is dysregulated, as it is in severe COVID-19 disease or other infectious or inflammatory diseases.

Effects of Dibutyltin Exposures on Translation Regulatory Factors eIF4E, eIF4B, and S6 in Human Immune Cells

Dibutyltin (DBT) is an organotin that contaminates the environment due to its uses as a stabilizer in polyvinylchloride, PVC plastics and as a deworming agent in certain poultry products. DBT has been found in drinking water and other beverages due to leaching from PVC plastics used during the distribution of drinking water and storage of beverages such as beers and wines. DBT has been found in human blood at levels as high as 0.3μM. Inflammatory cytokines are key mediators in the response to injury or infection. However, if their levels are increased in the absence of a necessary immune response, chronic inflammation is possible. Chronic inflammation is associated with various disorders including, rheumatoid arthritis, Crohn’s disease, atherosclerosis, and cancer. DBT can increase the production of pro‐inflammatory cytokines such as interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), interleukin 1 beta (IL‐1β), and interleukin 6 (IL‐6) in human immune cells. DBT appears to utilize the ERK 1/2 and/or p38 MAPK pathways to stimulate pro‐inflammatory cytokine production in immune cells. MAPK pathways are capable of regulating translation including processes leading to the phosphorylation (activation) of eukaryotic initiation factor 4E (eIF4E), eIF4B, and the S6 ribosomal subunit. The current study examines the levels and phosphorylation state of eIF4E, eIF4B and S6 after 6‐hour and 24‐hour exposures to DBT in peripheral blood mononuclear cells (PBMCs). DBT (at all concentrations) caused a robust increase in phosphorylation (activation) of eIF4B (S406) within 6‐hour exposure across all donors. Additionally, elevated levels of phosphorylated and total S6 were observed at 0.1 μM in all donors in 6‐hour exposures. At 24 hours of exposure, increases in P‐S6, S6 and P‐eIF4B (S406) begin to taper off with increases observed sporadically amongst donors. These results suggest that DBT may be in part elevating the production of key pro‐inflammatory cytokines in immune cells by its ability to activate translation.

A novel therapeutic vaccine based on graphene oxide nanocomposite for tumor immunotherapy

With an intensive understanding of the mechanism of immune system, developing a therapeutic tumor vaccine is one of the most perspective strategy of cancer immunotherapy. In this study, we report a facile approach to prepare graphene oxide (GO)-based therapeutic cancer-nanovaccine. The model antigen (ovalbumin, OVA) and adjuvant (CpG ODN), are conjugated with GO-PEI nanosheet through electrostatic interaction. The addition of PEG can improve biocompatibility and prevent nanoparticle aggregation. The prepared GO-based nanovaccine, GO-PEI-OVA-PEG-CpG, exhibits good biocompatibility and low toxicity both in vivo and in vitro. More importantly, it can efficiently induce the maturation of dendritic cells (DCs), the enhancement of antigen cross-presentation ability, and the amplification of cytokine production of immune cells. Impressively, this nanovaccine shows a remarkable therapeutic effect against pre-established B16-OVA-melanoma tumors, which can significantly inhibit tumor growth and prolong the survival time of the OVA-expressed tumor-bearing mice. Moreover, combining GO-PEI-OVA-PEG-CpG with NLG919, an IDO-1 (indoleamine-2,3-dioxygenase) inhibitor which can regulate the tumor microenvironment, displays a synergistic therapeutic effect. These findings indicate the GO-PEI-OVA-PEG-CpG nanovaccine actively induces an antigen-specific antitumor immune response and it combined with NLG919 could achieve better therapeutic outcomes.

RAMP1シグナルは，サイトカイン産生と好中球誘導を阻害することで急性肺傷害(ALI)を抑制する

Neuroimmune interactions have emerged as critical regulators of inflammation. Neuropeptide calcitonin gene-related peptide (CGRP) regulates cytokine production in immune cells through signaling for CGRP receptor, receptor activity-modifying protein 1 (RAMP1). Here, we examined the role of RAMP1 signaling in LPS-induced lung injury. Acute lung injury was induced by intratracheal injection of LPS in wild-type mice (WT) and RAMP1 knockout mice (RAMP1 KO). Compared with WT, RAMP1 KO exhibited decreases in survival rate and increases in lung injury score, and total protein concentrations and pro-inflammatory mediators including IL-6 and CXCL2 in BALF at 72 h after LPS administration. In WT, CGRP and RAMP1 levels in the lung were increased after LPS administration, and RAMP1 was expressed in alveolar macrophages. After LPS administration, the numbers of alveolar macrophages in both types of mice were diminished, and reached nadir at 6h, and restored to half levels of pre-values in WT thereafter, but remained low in RAMP1 KO until up to 72 h. By contrast, the numbers of neutrophils were increasing with time after LPS administration in two genotypes, and those in RAMP1 KO were larger than WT at 72 h. These results suggested that RAMP1 signaling attenuated LPS-induced acute lung injury by inhibiting cytokine production, neutrophil accumulation, and pulmonary vascular permeability.

Characterizing and overcoming innate immunity in beta-cell replacement therapy

In clinical islet transplantation, the engrafted islet cell mass is a critical factor for maintaining graft function and transplant outcome. This review dissects components that contribute to an acute innate immune response, including ischemia-reperfusion injury, damage-associated molecular patterns, islet-derived cytokines (isletokines), and an instant blood-mediated inflammatory reaction. The cyclical amplification of immune cell response and islet inflammation can occur at each stage of the transplant process. As such, strategies to improve islet transplantation include minimizing islet and immune cell cytokine production prior to procurement; suppressing the pretransplant islet immune inflammatory response; inhibiting the instant blood-mediated inflammatory reaction in the liver; preventing an innate immune response by encapsulation and islet surface modification; targeting cytokine/chemokine secretion and innate immune cell infiltration; considering the optimal site for islet engraftment; blocking cytokine signaling in islets; and implementing anti-inflammatory strategies to improve islet transplantation. A large body of evidence indicates that targeting immune components at multiple steps throughout the transplant procedure can improve overall islet transplant outcomes.

Bacterial Lipoteichoic Acid Attenuates Toll-Like Receptor Dependent Dendritic Cells Activation and Inflammatory Response.

Toll-like receptor (TLR) signaling is an indispensable factor in immune cells activation. Many TLR ligands have been identified, and were characterized the immunological functions such as inflammatory cytokine production in immune cells. However, the anti-inflammatory response in TLR ligand-mediated manner is poorly understood. In this report, we show that bacterial lipoteichoic acid (LTA), which is a TLR2 ligand from gram-positive bacteria including Staphylococcus aureus (S. aureus), suppresses TLR-mediated inflammatory response in dendritic cells (DCs). The TLR ligand-induced Tumor Necrosis Factor-alpha (TNF-α) production was suppressed in the bone marrow derived dendritic cells (BMDCs) by co-treatment of LTA. The cellular activation, which was characterized as upregulations of CD80, CD86 and major histocompatibility complex II (MHC II) expression, was also suppressed in the TLR ligand stimulated BMDCs in the presence of LTA. While LTA itself didn’t induced both TNF-α production and upregulation of cell surface markers. The LTA mediated immunosuppressive function was abolished by TLR2 blocking in lipopolysaccharide (LPS)-stimulated BMDCs. Furthermore, LTA also showed the immunosuppressive function in the generation of IFN-γ+CD4+ T (Th1) cells by attenuation of antigen presenting activity in the BMDCs. In the imiquimod (IMQ)-induced acute skin inflammation, LTA suppressed the inflammation by downregulation of the activation in skin accumulated DCs. Thus, LTA is a TLR2 dependent immunological suppressor against inflammatory response induced by other TLR ligands in the DCs.

Role of prostaglandins in rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease characterised by systemic and chronic synovitis that lead to joint destruction, pain, and many complications. Treatments only relieve certain symptoms, but do not cure RA completely. Prostaglandins (PGs) are lipid signalling molecules and released in the early phase of RA. Increasing evidences have shown five main contribution of PGs to the different stages and symptoms of RA. First, PGs maintain the autoimmune response and immune-system inflammation by modulating the differentiation, maturation, and cytokine production of immune cells. Second, PGs are beneficial for leukocyte infiltration, synovial hyperplasia, and angiogenesis to promote synovitis. Third, PGs are involved in cartilage degradation and bone resorption. Fourth, PGs are important mediators of joint-pain regulation. Finally, in the late stage of RA inflammation, PGs play a part in joint protection. Those findings suggest that PGs are potential therapy targets for RA. This review highlights recent advances in the RA development caused by PGs, and provides recommendations for future research directions.

New Zealand Blackcurrant Extract Increases Circulating Hsp32 And Hsp90α But Doesn’T Affect Circulating Hsp72

Extracellular heat shock protein 72 (eHSP72) acts as an inflammatory molecule, inducing cytokine production in immune cells, whereas HSP90α is implicit in recovery and adaptation to cellular stress. Heme oxygenase-1 (eHSP32) protects the vasculature and suppresses inflammation. Each are elevated following exertional heat stress. Polyphenols are proposed to have anti-inflammatory properties, so may affect eHSP responses to exercise. PURPOSE: To determine the effects of 7-days supplementation with New Zealand blackcurrant (NZBC) extract on eHSP72, eHSP90α, and eHSP32 before and after exertional heat stress. METHODS: In a randomized double-blind design, 12 men (Age: 28±6 years, stature: 1.81±0.07 m, mass: 80.5±9.8 kg, VO2max: 55.6±6.0 mL.kg-1.min-1) completed 2 trials. Participants ingested 2x300 mg.day-1 capsules of CurraNZ™ (each containing 105 mg anthocyanin) or a visually matched placebo for 7 days (washout 14 days). On day 7, participants ran 60 minutes at 65%VO2max in hot ambient conditions (34°C and 40% relative humidity). eHSP72, eHSP90a, and eHSP32 were measured in EDTA plasma at rest and 20 and 60 minutes after exercise. RESULTS: Post exercise eHSP72 concentrations were elevated after the placebo [by 1.98 ng.mL-1 (95% CI: 0.65 - 3.33 ng.mL-1)] and NZBC trials [by 1.59 ng.mL-1 (95% CI: 0.03 - 3.15 ng.mL-1)] and remained elevated 60 minutes after exercise [Placebo: by 0.68 ng.mL-1 (95% CI: -0.07 - 1.46 ng.mL-1); NZBC by 0.51 ng.mL-1 (95% CI: -0.37 - 1.40 ng.mL-1)]. Basal eHSP90α concentration was increased following NZBC supplementation [by 5.60 ng.mL-1 (1.85 - 9.51 ng.mL-1), trial x time interaction, F = 3.57, p = 0.046, np2 = 0.25), and were elevated at 20 and 60 minutes post exercise in both conditions. Similarly, basal eHSP32 was elevated after NZBC supplementation [by 3.9 ng.mL-1 (95% CI: 0.37 - 7.42 ng.mL-1), trial x time interaction F = 5.62, p = 0.01, np2 = 0.34), but were not altered at 20 or 60 minutes after heat stress in either condition. CONCLUSION: We present moderate evidence to support that 7 days of NZBC extract supplementation increases basal eHSP32 and eHSP90a, with no effect on eHSP72 before or after exercise. Further research is required to determine the functional relevance of these increases. Acknowledgement: We thank Health Currancy Ltd (UK) and CurraNZ (NZ) for supplements.

Nonspecific effects of oral vaccination with live-attenuated Salmonella Typhi strain Ty21a.

Epidemiological and immunological evidence suggests that some vaccines can reduce all-cause mortality through nonspecific changes made to innate immune cells. Here, we present the first data to describe the nonspecific immunological impact of oral vaccination with live-attenuated Salmonella Typhi strain Ty21a. We vaccinated healthy adults with Ty21a and assessed aspects of innate and adaptive immunity over the course of 6 months. Changes to monocyte phenotype/function were observed for at least 3 months. Changes to innate and adaptive immune cell cytokine production in response to stimulation with vaccine and unrelated nonvaccine antigens were observed over the 6-month study period. The changes that we have observed could influence susceptibility to infection through altered immune responses mounted to subsequently encountered pathogens. These changes could influence all-cause mortality.

Synergistic Effects of Weightlessness, Isoproterenol, and Radiation on DNA Damage Response and Cytokine Production in Immune Cells.

The implementation of rotating-wall vessels (RWVs) for studying the effect of lack of gravity has attracted attention, especially in the fields of stem cells, tissue regeneration, and cancer research. Immune cells incubated in RWVs exhibit several features of immunosuppression including impaired leukocyte proliferation, cytokine responses, and antibody production. Interestingly, stress hormones influence cellular immune pathways affected by microgravity, such as cell proliferation, apoptosis, DNA repair, and T cell activation. These pathways are crucial defense mechanisms that protect the cell from toxins, pathogens, and radiation. Despite the importance of the adrenergic receptor in regulating the immune system, the effect of microgravity on the adrenergic system has been poorly studied. Thus, we elected to investigate the synergistic effects of isoproterenol (a sympathomimetic drug), radiation, and microgravity in nonstimulated immune cells. Peripheral blood mononuclear cells were treated with the sympathomimetic drug isoproterenol, exposed to 0.8 or 2 Gy γ-radiation, and incubated in RWVs. Mixed model regression analyses showed significant synergistic effects on the expression of the β2-adrenergic receptor gene (ADRB2). Radiation alone increased ADRB2 expression, and cells incubated in microgravity had more DNA strand breaks than cells incubated in normal gravity. We observed radiation-induced cytokine production only in microgravity. Prior treatment with isoproterenol clearly prevents most of the microgravity-mediated effects. RWVs may be a useful tool to provide insight into novel regulatory pathways, providing benefit not only to astronauts but also to patients suffering from immune disorders or undergoing radiotherapy.

Crystal structure of the ligand-free form of the Vps10 ectodomain of dimerized Sortilin at acidic pH.

Sortilin is a multifunctional sorting receptor involved in cytokine production in immune cells. To understand the mechanism of Sortilin-mediated cytokine trafficking, we determined the 2.45-Å structure of the dimerized Sortilin ectodomain (sSortilin or the Vps10-domain) crystallized at acidic pH. Substantial conformational changes upon dimerization lead to the intermolecular hydrophobic interaction between the conserved E455 and F137. Analysis of the electrostatic surface and size-exclusion chromatography revealed that sSortilin dimerization occurs due to an increase in hydrophobic interactions at the neutral dimer interface at acidic pH. The N682-attached N-glycan in the vicinity of the dimer interface implies its involvement in the dimerization. The disruption of Sortilin dimerization by mutations impairs efficient interferon-alpha secretion from cells. These results suggest the functional importance of Sortilin dimerization in cytokine trafficking.

Guanabenz inhibits TLR9 signaling through a pathway that is independent of eIF2α dephosphorylation by the GADD34/PP1c complex.

Endoplasmic reticulum (ER) stress triggers or amplifies inflammatory signals and cytokine production in immune cells. Upon the resolution of ER stress, the inducible phosphatase 1 cofactor GADD34 promotes the dephosphorylation of the initiation factor eIF2α, thereby enabling protein translation to resume. Several aminoguanidine compounds, such as guanabenz, perturb the eIF2α phosphorylation-dephosphorylation cycle and protect different cell or tissue types from protein misfolding and degeneration. We investigated how pharmacological interference with the eIF2α pathway could be beneficial to treat autoinflammatory diseases dependent on proinflammatory cytokines and type I interferons (IFNs), the production of which is regulated by GADD34 in dendritic cells (DCs). In mouse and human DCs and B cells, guanabenz prevented the activation of Toll-like receptor 9 (TLR9) by CpG oligodeoxynucleotides or DNA-immunoglobulin complexes in endosomes. In vivo, guanabenz protected mice from CpG oligonucleotide-dependent cytokine shock and decreased autoimmune symptom severity in a chemically induced model of systemic lupus erythematosus. However, we found that guanabenz exerted its inhibitory effect independently of GADD34 activity on eIF2α and instead decreased the abundance of CH25H, a cholesterol hydroxylase linked to antiviral immunity. Our results therefore suggest that guanabenz and similar compounds could be used to treat type I IFN-dependent pathologies and that CH25H could be a therapeutic target to control these diseases.

Lactobacillus acidophilus modulates inflammatory activity by regulating the TLR4 and NF-κB expression in porcine peripheral blood mononuclear cells after lipopolysaccharide challenge.

A total of forty weaned pigs ((Landrace × Yorkshire) × Duroc) were used to evaluate the effects of Lactobacillus acidophilus on inflammatory activity after lipopolysaccharide (LPS) challenge. Experimental treatments were as follows: (T1) control diet+saline challenge; (T2) control diet with 0·1% L. acidophilus+saline challenge; (T3) control diet+LPS challenge; and (T4) control diet with 0·1% L. acidophilus+LPS challenge. On d-14, piglets were challenged with saline (T1 and T2) or LPS (T3 and T4). Blood samples were obtained at 0, 2, 4, 6 and 12 h after being challenged and analysed for immune cell cytokine production and gene expression pattern. The L. acidophilus treatment increased the average daily weight gain (ADWG) and average daily feed intake (ADFI) compared with the control diet. With the control diet, the LPS challenge (T3) increased the number of immune cells and expression of TNF-α and IL-6 compared with the saline challenge (T1). Whereas with the saline challenge L. acidophilus treatment (T2) increased the number of leucocytes and CD4 compared with the control diet (T1), with the LPS challenge L. acidophilus treatment (T4) decreased the number of leucocytes, lymphocytes, CD4+ and CD8+ and expression of TNF-α and IL-6 compared with the control diet (T3). L. acidophilus treatment decreased the expression of TRL4 and NF-κB in peripheral blood mononuclear cells (PBMC) after LPS challenge, which leads to inhibition of TNF-α, IFN-γ, IL-6, IL-8 and IL1B1 and to induction of IL-4 and IL-10. We suggested that L. acidophilus improved ADWG and ADFI and protected against LPS-induced inflammatory responses by regulating TLR4 and NF-κB expression in porcine PBMC.

Screening of Immune-Active Lactic Acid Bacteria

The purpose of this study was to investigate the effect of lactic acid bacteria (LAB) cell wall extract on the proliferation and cytokine production of immune cells to select suitable probiotics for space food. Ten strains of LAB (Lactobacillus bulgaricus, L. paracasei, L. casei, L. acidophilus, L. plantarum, L. delbruekii, Lactococcus lactis, Streptococcus thermophilus, Bifidobacterium breve, and Pedicoccus pentosaceus) were sub-cultured and further cultured for 3 d to reach 7-10 Log colony-forming units (CFU)/mL prior to cell wall extractions. All LAB cell wall extracts failed to inhibit the proliferation of BALB/c mouse splenocytes or mesenteric lymphocytes. Most LAB cell wall extracts except those of L. plantarum and L. delbrueckii induced the proliferation of both immune cells at tested concentrations. In addition, the production of TH1 cytokine (IFN-γ) rather than that of TH2 cytokine (IL-4) was enhanced by LAB cell wall extracts. Of ten LAB extracts, four (from L. acidophilus, L. bulgaricus, L. casei, and S. thermophiles) promoted both cell proliferating and TH1 cytokine production. These results suggested that these LAB could be used as probiotics to maintain immunity and homeostasis for astronauts in extreme space environment and for general people in normal life.

Lactobacillus helveticus SBT2171, a cheese starter, regulates proliferation and cytokine production of immune cells

Consumption of a Lactobacillus helveticus SBT2171 (LH2171)-containing cheese has been reported to exhibit immunoregulatory actions, including an increase in regulatory T cell population and reduction in proinflammatory cytokine production in mice. We examined the in vitro effects of LH2171 cells per se on immune cell function, specifically proliferation and cytokine production, which are primary reactions of the immune response. Immune cell fractions were prepared by mechanical disruption of mesenteric lymph nodes (MLN), Peyer’s patches (PP), and spleens (SP) of mice. The cell fractions were dispensed into a culture plate and stimulated with anti-CD3/CD28 antibody beads in place of antigen-presenting cells or lipopolysaccharide (LPS) in the presence or absence of heat-treated LH2171 cells and other bacterial strains for comparison. After incubation, proliferation, cytokine production, and cell viability of the immune cells were determined. The LH2171 significantly inhibited the proliferation of MLN immune cells stimulated with anti-CD3/CD28 compared with other bacterial strains. The antiproliferative potency of LH2171 was effective not only on MLN but also on PP and SP stimulated with anti-CD3/CD28 or LPS. The LH2171 also decreased LPS-stimulated IL-6 production from MLN, PP, and SP, and IL-1β production from SP, but LH2171 did not affect the viability of immune cells. The LH2171 inhibited immune cell proliferation and proinflammatory cytokine (IL-6 and IL-1β) production. The inhibitory actions were not due to cytotoxicity to immune cells, suggesting that LH2171 is a dairy Lactobacillus strain with beneficial immunoregulatory properties.

Effect of the Premalignant and Tumor Microenvironment on Immune Cell Cytokine Production in Head and Neck Cancer

Head and neck squamous cell carcinoma (HNSCC) is marked by immunosuppression, a state in which the established tumor escapes immune attack. However, the impact of the premalignant and tumor microenvironments on immune reactivity has yet to be elucidated. The purpose of this study was to determine how soluble mediators from cells established from carcinogen-induced oral premalignant lesions and HNSCC modulate immune cell cytokine production. It was found that premalignant cells secrete significantly increased levels of G-CSF, RANTES, MCP-1, and PGE2 compared to HNSCC cells. Splenocytes incubated with premalignant supernatant secreted significantly increased levels of Th1-, Th2-, and Th17-associated cytokines compared to splenocytes incubated with HNSCC supernatant. These studies demonstrate that whereas the premalignant microenvironment elicits proinflammatory cytokine production, the tumor microenvironment is significantly less immune stimulatory and may contribute to immunosuppression in established HNSCC.