IL-6 In Supernatants Research Articles

Candida Administration in 5/6 Nephrectomized Mice Enhanced Fibrosis in Internal Organs: An Impact of Lipopolysaccharide and (1→3)-β-D-Glucan from Leaky Gut.

Uremic toxins and gut dysbiosis in advanced chronic kidney disease (CKD) can induce gut leakage, causing the translocation of gut microbial molecules into the systemic circulation. Lipopolysaccharide (LPS) and (1→3)-β-D-glucan (BG) are the major gut microbial molecules of Gram-negative bacteria and fungi, respectively, and can induce inflammation in several organs. Here, the fibrosis in the kidney, liver, and heart was investigated in oral C. albicans-administered 5/6 nephrectomized (Candida-5/6 Nx) mice. At 20 weeks post 5/6 Nx, Candida-5/6 Nx mice demonstrated increased 24 h proteinuria, liver enzymes, and serum cytokines (TNF-α, IL-6, and IL-10), but not weight loss, systolic blood pressure, hematocrit, serum creatinine, or gut-derived uremic toxins (TMAO and indoxyl sulfate), compared to in 5/6 Nx alone. The gut leakage in Candida-5/6 Nx was more severe, as indicated by FITC-dextran assay, endotoxemia, and serum BG. The areas of fibrosis from histopathology, along with the upregulated gene expression of Toll-like receptor 4 (TLR-4) and Dectin-1, the receptors for LPS and BG, respectively, were higher in the kidney, liver, and heart. In vitro, LPS combined with BG increased the supernatant IL-6 and TNF-α, upregulated the genes of pro-inflammation and pro-fibrotic processes, Dectin-1, and TLR-4 in renal tubular (HK-2) cells and hepatocytes (HepG2), when compared with LPS or BG alone. This supported the pro-inflammation-induced fibrosis and the possible LPS-BG additive effects on kidney and liver fibrosis. In conclusion, uremia-induced leaky gut causes the translocation of gut LPS and BG into circulation, which activates the pro-inflammatory and pro-fibrotic pathways, causing internal organ fibrosis. Our results support the crosstalk among several organs in CKD through a leaky gut.

Establishment of local allergic rhinitis tolerance in mouse model

Objective:To establish a mouse model of local allergic rhinitis tolerance by intranasal infusion of allergen, and study its immunological indexes and characteristics. Methods:The mice were given ovalbumin（OVA） and phosphate buffer solution（PBS） daily, and their allergic symptoms were recorded. OVA-specific antibodies（IgE, IgG1, IgG2a） in serum and cytokine（IL-4, IL-10, IFN-γ） in splenic culture supernatant were detected. The infiltration of eosinophils and goblet cells in nasal mucosa were observed, and the changes in local nasal mucosa genes were analyzed by RNA-seq technique. Results:Mice first showed aggravation of allergic symptoms when stimulated with OVA. The serum OVA-specific antibodies IgE, IgG1, IgG2a and the cytokine（IL-4, IL-10, IFN-γ） Iin splenic culture supernatant were increased, the eosinophils and goblet cells in nasal mucosa were significantly increased. The expression of encoding IL-10, TGF-β gene and eosinophils activation gene in nasal mucosa were up-regulated. As the duration of nasal dripping increased, the allergic symptoms gradually decreased, serum OVA-specific antibodies IgE, IgG1, IgG2a continued to increase. Splenic culture supernatant IL-4 decreased, IL-10 increased, IFN-γ had a downward trend. In nasal mucosa, goblet cells decreased significantly. Genes involved in eosinophils activation were significantly down-regulated. The encoding tolerance genes such as IL-10 and TGF-β genes remained highly expressed. Ten core genes associated with immune tolerance in localized allergic rhinitis were screened, Rps27a, Uba52, Kng2, Gnal, C3, Rtp4, Reep1, Rtp2, Rtp1, Reep5. Conclusion:The mice first develop an allergy and then develop immune tolerance by continuous local drops of the allergen. The generation of immune tolerance may be induced by the continuous action of allergens, which induced systemic and local immune tolerance effects.

Infusion of Cultured Endothelial Progenitor Cells Boosts Hematopoietic Recovery and Increases the Expression of LepR+ Cells after Irradiation

The hemopoietic system's function of producing blood cells and immune cells during thorough life relies on the Maintainance and differentiation of hematopoietic stem cells (HSCs). HSCs activities were tightly and finely tuned by a specialized microenvironment called "niche" in the bone marrow. However, during the routine treatment of hematopoietic diseases, such as chemotherapy and radiotherapy, the niche was inevitably impaired which is one of the essential causes of hematopoietic suppression. Our previous research results showed that infusion of cultured endothelial progenitor cells (EPCs) could boost hematopoietic recovery after HSCT. And the latent mechanisms could be that EPCs can differentiate into endothelial cells (ECs) in vivo. However, the roles of EPCs play in the bone marrow niche and the specific mechanism remain unknown. Here we designed further to investigate the effect and mechanism of EPCs in boosting bone marrow microenvironment recovery after total body irradiation (TBI). This may offer a new cue for therapy of hematopoietic suppression after the treatment of malignant hematological diseases. Bone marrow-derived Primary EPCs isolation and culture were described as before. Briefly, Bone marrow cells from Balb/c or C57BL/6J mice aged 6-8 weeks were cultured with EGM-2 medium which was changed in 36 h, the adherent cells formed colonies identified as elongated sprouting cells radiating from a central core of round cells and identified as VEGFR2+; CD133+; CD144+; SCA1+ by FACS on days 4-6. To assess the effects of the EPCs in boosting hematopoiesis, Balb/c mice underwent TBI with a lethal dose and were injected withEPCs or the same volume of PBS via tail vein in 4-6 hours. The survival of the EPC group was elevated ( survival: 8/9 vs 0/13; p＜0.001). C57BL/6J mice underwent TBI with a sublethal dose and were injected with the cultured EPCs isolated from C57BL/6-Tg(CAG-EGFP) mice or the same volume of PBS via tail vein in 4-6 hours. Results showed that EPCs boost the recovery of hematopoietic cells in the bone marrow and peripheral blood cells; Immunofluence (IF) staining showed that Infused EPCs with GFP fluorescence were home to bone marrow and differentiated into ECs which were marked with endomucin (EMCN); Moreover, assessments of the alteration of bone marrow niche using H&E staining revealed that infusion of EPCs reduced the accumulation of fat and using multi-analyte flow assay showed that mice infused with EPCs express a higher level of M-CSF and TGF-β in plasma while the level of SCF and IL-6 both in plasma and bone marrow supernatant were lower compared to TBI. Furthermore, to assess the change of sinusoidal niche, we used IF staining and found that the sinusoids were dilated and perisinusoidal stromal cells identified as LepR+ were massively lost on the 7th day after TBI while the dilated sinusoidal lumens were narrowed (11.05±1.789 vs 20.86±4.573，p=0.0258) and LepR+ cells were upregulated on the 21st day after the infusion of EPCs which indicated that the sinusoidal niche has a better recovery compared to TBI. In Conclusion, EPCs could differentiate into endothelial cells in vivo boosting the recovery of the hematopoietic system. And the potential mechanism is that EPCs repair the bone marrow sinusoidal niche damaged by irradiation.

Cl-amidine attenuates lipopolysaccharide-induced inflammation in human gingival fibroblasts via the JNK/MAPK, NF-κB, and Nrf2 signalling pathways.

Cl-amidine has been reported to have anti-inflammatory properties in a variety of diseases. However, the role of Cl-amidine in periodontal disease remains unclear. Here, the purpose of this study was to investigate the effect of Cl-amidine on lipopolysaccharide (LPS)-induced inflammation in human gingival fibroblasts (HGFs). The cytotoxic effect of Cl-amidine was measured with the Cell Counting Kit-8 (CCK-8) assay and Annexin V-FITC/PI staining. The protein levels of IL-6 and IL-8 in culture supernatants were measured with enzyme-linked immunosorbent assay (ELISA). The mRNA levels of inflammatory cytokines, TLR4 and MyD88 were assessed by quantitative real-time polymerase chain reaction (qRT-PCR) analysis. The expression patterns of IL-6, TNF-ɑ, and IL-1β in HGFs were tested with western blot. The levels of NF-κB, MAPK, and Nrf2 pathway-related proteins were detected by western blot. Immunofluorescence (IF) staining was used to examine the nuclear translocation of NF-κB p65. Moreover, a rat gingivitis model was established to further clarify the role of Cl-amidine. Our results showed that Cl-amidine suppressed LPS-induced gingival inflammation both in vitro and in vivo. Mechanistically, Cl-amidine inhibited LPS-induced MyD88 expression, NF-κB activation, and JNK phosphorylation. Additionally, Cl-amidine upregulated Nrf2 and Ho-1 expression both with and without LPS stimulation but did not alter ROS levels or Keap1 expression. Overall, our data suggest that Cl-amidine acts as an inhibitor of LPS-induced gingival inflammation via the JNK/MAPK, NF-κB, and Nrf2 signalling pathways.

Zinc Oxide Tetrapods Modulate Wound Healing and Cytokine Release In Vitro-A New Antiproliferative Substance in Glaucoma Filtering Surgery.

Glaucoma filtering surgery is applied to reduce intraocular pressure (IOP) in cases of uncontrolled glaucoma. However, postoperative fibrosis reduces the long-term success of both standard trabeculectomy and microstents. The aim of this study was to test the antiproliferative and anti-inflammatory potential of ZnO-tetrapods (ZnO-T) on human Tenon's fibroblasts (HTFs) for glaucoma surgery. The toxicity of ZnO-T on HTFs was determined using an MTT test. For analysis of fibroblast proliferation, migration, and transdifferentiation, cultures were stained for Ki67, alpha-smooth muscle actin (α-SMA), and p-SMAD. A fully quantitative multiplex ELISA was used to determine the concentrations of different cytokines, platelet-derived growth factor (PDGF), and hepatocyte growth factor (HGF) in culture supernatants with and without previous ZnO-T treatment. Treatment with higher concentrations (10 and 20 µg/mL) was associated with HTF toxicity, as shown in the wound healing assay. Furthermore, the number of Ki67, α-SMA-positive, and pSMAD-positive cells, as well as IL-6 and HGF in supernatants, were significantly reduced following incubation with ZnO-T. In conclusion, we were able to show the antiproliferative and anti-inflammatory potentials of ZnO-T. Therefore, the use of ZnO-T may provide a new approach to reducing postoperative fibrosis in glaucoma filtering surgery.

Discovery of benzimidazole substituted 1, 2, 4-oxadiazole compounds as novel anti-HBV agents with TLR8-agonistic activities

Hepatitis B virus (HBV) infection is a public health threat worldwide and characterized by a dysfunctional immune response. In the present work, a new series of benzimidazole substituted 1, 2, 4-oxadiazole compounds were designed as immunomodulatory anti-HBV agents. Data showed compound 11o displayed significant in vitro anti-HBV activities against wild-type and nucleos(t)ide analogues-resistant HBV with IC50 values of 0.53 and 0.44 μM, respectively. In contrast, nucleos(t)ide analogue lamivudine is only effective for wild-type HBV (IC50 < 0.1 μM) but not effective for resistant HBV (IC50 > 100 μM). Dual-luciferase reporter gene and ELISA assay revealed that 11o exhibited a dose-dependent effect on inducing TLR8-regulated NF-κB activity, and could promote the secretion of cytokines TNF-α and IL-12 in supernatant from human PBMC cells. Molecular docking studies found that 11o formed tight interactions with binding pocket residues located at the dimer interface of TLR8. Considering the potent in vitro anti-HBV activity, effective TLR8-agonistic potency, and relatively safe profile with a selectivity index (SI) value high above 37, compound 11o deserves further investigation as a potential immunomodulatory anti-HBV agent.

Oxaliplatin Induces Immunogenic Cell Death in Human and Murine Laryngeal Cancer

Background Cisplatin resistance is observed in patients with laryngeal cancer. The present study was designed to explore the efficacy of oxaliplatin on laryngeal cancer and elucidate the underlying mechanisms. Methods Cell viability was determined by using MTT assays. Cell apoptosis was determined by using annexin V and propidium iodide (PI) staining. Flow cytometry and immunofluorescence were applied to determine the levels of calreticulin (CALR) and DiD (1,1-dioctadecyl-3,3,3,3-tetramethylindodicarbocyanine). Flow cytometry was applied to analyze the levels of CD83, CD86, IFN-γ-producing CD8+ T cells, and CD4+CD25+FoxP3+ Tregs. The levels of adenosine triphosphate (ATP) were determined by using a chemiluminescent ATP kit and cytokines were determined by using specific enzyme-linked immunosorbent assays (ELISAs). The levels of HMGB1 were determined by using Western blot and ELISA, respectively. The xenograft animal model was constructed to evaluate the antitumor effects of oxaliplatin. Results Oxaliplatin inhibited cell growth, promoted cell apoptosis, and induced the levels of CALR, ATP, and high mobility group box protein 1 (HMGB1) in Hep-2 cells. Oxaliplatin-treated Hep-2 cells increased the intensity of DiD and the levels of CD83 and CD86 in dendritic cells (DCs), as well as induced the supernatant IL-6 and TNF-α. Oxaliplatin-treated primary laryngeal cancer cell-pulsed DCs increased the IFN-γ-producing CD8+ T cells and suppressed CD4+CD25+FoxP3+ Tregs. In vivo data showed that oxaliplatin suppressed tumor growth and increased the populations of CD86+CD80+ and CD8+CD45+ cells in the tumor tissues. Conclusion Treatment with oxaliplatin inhibited laryngeal cancer cells by inducing immunogenic cell death.

MiR-135a is highly expressed and aggravates inflammatory response in sepsis by targeting MYOM1.

Our research attempted to explore the effect of miR-135a on lipopolysaccharide (LPS)-induced THP-1cells damage and its potential mechanism. LPS (1 μg/mL) was selected to mimic the injury of sepsis in vitro. qRT-PCR was used to detect miR-135a expression. The association between miR-135a and Myomesin 1 (MYOM1) was speculated bythe predication website and confirmed by the dual-luciferase assay. MYOM1 expression was observed by qRT-PCR and western blotting assays. The biological properties of THP-1cells were analyzed by cell counting kit-8 and flow cytometry assays. The concentration of TNF-α, IL-6 and IL-8 in cell supernatant was calculated by enzyme-linkedimmunosorbentassay. A marked augmentation of miR-135a was observed in LPS-induced THP-1 cells. Moreover, depletion of miR-135a alleviated the LPS-induced THP-1 cells injury from the perspective of improving cell viability and reducing cell apoptosis. Importantly, MYOM1, which was under expressed in LPS-induced THP-1 cells, was identified as a target of miR-135a and negatively regulated by miR-135a. Additionally, the mitigative impact of miR-135a inhibitor on THP-1cells damage triggered by LPS was suppressed by MYOM1 depletion. Our study suggested that the protective effect of miR-135a inhibitor on LPS-induced THP-1 cells injury was realized by regulation of MYOM1, which might afford a pair of novel molecules for sepsis clinical diagnosis.

MiR-128-3p inhibits high-glucose-induced peritoneal mesothelial cells fibrosis via PAK2/SyK/TGF-β1 axis.

To elucidate the mechanism of miR-128-3p in peritoneal fibrosis (PF). Peritoneal mesothelial cells (PMCs) were dealt with high glucose (HG) for 3 days. The expressions of miR-128-3p, p21-activated kinase 2 (PAK2), spleen tyrosine kinase (SyK), and transforming growth factor-β1 (TGF-β1) were detected with quantitative real-time reverse transcription polymerase chain reaction. The levels of IL-1β, TNF-α, IL-6, and monocyte chemotactic protein-1 in supernatant were measured by ELISA. Proteins of TGF-β1, SyK, PAK2, α-SMA, collagen I, vimentin, ERK/AP-1, and IκBα/NF-κB pathway related proteins were measured by Western blot. The correlation between miR-128-3p and PAK2 was found by bioinformatics analysis and luciferase reporter gene analysis. miR-128-3p was decreased while PAK2, SyK, and TGF-β1 were increased in HG-induced PMCs. Moreover, miR-128-3p inhibited HG-induced fibrosis and inflammation in PMCs by targeting PAK2. PAK2 activated SyK, which induced TGF-β1 expression through ERK/AP-1 and IκBα/NF-κB pathways to promote HG-induced fibrosis of PMCs. miR-128-3p inhibited HG-induced PMCs fibrosis via PAK2/SyK/TGF-β1 axis.

Effect of Xijiao Dihuang Combined Prescription on Human Dendritic Cell Function Induced by Lipopolysaccharide

To observe the effects of drug-containing serum of Xijiao Dihuang combined prescription(XJDH) on the related functions of dendritic cells(DCs) induced in vitro, and to explore the mechanisms underlying the effectiveness of XJDH treatment on primary immune thrombocytopenia(ITP). Peripheral blood samples were colle-ted from 6 healthy volunteers. Mononuclear cells were isolated by density gradient centrifugation, and CD14+ mononuclear cells were collected by the magnetic separation technique. CD14+ mononuclear cells were induced into immature DCs by recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) and recombinant human interleukin 4 (IL-4). Immature DCs were divided into three groups: control group, model group and XJDH group. CCK-8 assay was used to determine the intervention concentration and time of drug-containing serum. Lipopolysaccharide(LPS) with the final concentration of 1 μg/ml was added to model group and XJDH group respectively for 24 h to induce DCs maturation. Normal rat serum was added to control group and model group, and XJDH was added to XJDH group for 24 h. Flow cytometry was used to detect the levels of CD80, CD83 and HLA-DR on the surface of DCs. Western blot was used to detect the expression of TLR4 and NF-κB, and levels of IL-6, IL-12 and TNF-α in cell supernatant was detected by ELISA. Compared with the control group, LPS stimulation increased the expression of CD80, CD83 and HLA-DR, with subsequent increasing expression of TLR4 and NF-κB, as well as IL-6, IL-12 and TNF-α increased(P<0.05). In comparison with model group, the expression of DCs surface molecules CD80, CD83 and HLA-DR, DCs' expression of TLR4 and NF-κB protein, and the levels of IL-6, IL-12 and TNF-α in the cell supernatant of XJDH group decreased after the intervention of XJDH (P<0.05). Drug containing serum of Xijiao Dihuang combined prescription can down-regulate TLR4/NF-κB signaling pathway related protein expression, inhibit DCs maturation, and reduce proinflammatory factor secretion, which may be one of the mechanisms of drug-containing serum of Xijiao Dihuang combined prescription in the treatment of immune thrombocytopenia.

Downmodulation of Regulatory T Cells Producing TGF-β Participates in Pathogenesis of Leprosy Reactions.

Leprosy reactions are an acute and systemic manifestation, which occurs suddenly, can be severe and lead leprosy patients to disability. Reactional episodes are observed among half of the multibacillary patients, mainly in borderline lepromatous and lepromatous forms. They may begin at any time during multidrug therapy, and even before the treatment. Physical disabilities, which are the source of extreme suffering and pain for patients, occur in progression of the cellular immune response associated with a reaction and are still poorly understood. Thus, this work aimed to phenotypically and functionally characterize CD4+ and CD8+ Treg cells ex vivo and in response to Mycobacterium leprae (ML). We studied 52 individuals, including 18 newly diagnosed and untreated multibacillary leprosy patients, 19 reactional multibacillary patients (Type I or Type II episodes) and 15 healthy volunteers, included as controls, all residents of the city of Rio de Janeiro. The functional activity and frequencies of these cells were evaluated through multiparametric flow cytometry. In addition, the production of cytokines in supernatant from peripheral blood mononuclear cell cultures was also investigated against ML by enzyme-linked immunosorbent assay. Our results showed a decrease in CD4+TGF-β+ Treg and CD8+ TGF-β+ Treg in leprosy multibacillary patients during both types of reactional episodes. Alterations in the cytokine profile was also observed in Type II reactions, along with upregulation of IL-17 and IL-6 in supernatant. Thus, our study suggests that downregulation of Treg cells is related with both classes of reactional episodes, improving our understanding of immune hyporesponsiveness in multibacillary patients and hyperesponsiveness in both reactions.

Shikonin combined with methotrexate regulate macrophage polarization to treat psoriasis

ABSTRACT This study aimed to investigate whether shikonin combined with methotrexate could inhibit psoriasis progression by regulating the polarization of macrophages through in vivo and in vitro experiments. Imiquimod was administrated to the exposed skin of BALB/c mice, and shikonin and methotrexate suspension were also given by gavage. The erythema, scales and thickness were scored for mice lesions in each group, and the total score was obtained by adding the above three scores, and calculated as psoriasis area and severity index (PASI) score. The skin lesion tissue from mice was isolated and used for hematoxylin-eosin staining and immunohistochemistry assay. Drug-containing serum was prepared and administrated into mouse macrophage RAW264.7 cells, followed by simulation of LPS. The levels of tumor necrosis factor-α (TNF-α), Interleukin (IL)-1β, and IL-6 in cell supernatant were assessed using ELISA Kits and real-time PCR. In imiquimod-induced psoriasis mice, shikonin combined with methotrexate exerted protective effects by reducing erythema and PASI scores, decreasing backer score and epidermal thickness, and particularly regulating macrophage polarization. In LPS-stimulated RAW264.7 cells, shikonin combined with methotrexate regulated M1/M2 polarization and altered the levels of M1 markers. Shikonin combined with methotrexate inhibit psoriasis progression by regulating the polarization of macrophages, which may be useful in the treatment of psoriasis.

Novel Th17 Lymphocyte Populations, Th17.1 and PD1+Th17, are Increased in Takayasu Arteritis, and Both Th17 and Th17.1 Sub-Populations Associate with Active Disease.

PurposeWe evaluated T helper lymphocyte profile, including novel Th17 subsets Th17.1 (secrete IFN-γ, associate with corticosteroid resistance) and PD1+Th17 (secrete TGF-β1, implicated in fibrosis), and related cytokines in peripheral blood of Takayasu arteritis (TAK).Materials and MethodsWe evaluated circulating Th1, Th2, Th17, Th17.1, PD1+CD4+ T lymphocytes, PD1+Th17, and Treg lymphocytes, inflammatory (IFN-γ, IL-4, IL-6, IL-17A, IL-23, IL-1β, TNF-α) and regulatory (IL-10, TGF-β1) cytokines in peripheral blood of TAK (n = 57; median age 35 (interquartile range 26–45) years; 40 females) in a cross-sectional design. We studied inflammatory and regulatory cytokines in culture supernatant of peripheral blood mononuclear cells (PBMCs) from TAK following stimulation with anti-CD3/anti-CD28 and their modulation by tacrolimus (immunosuppressive) with/without tadalafil (anti-fibrotic). Furthermore, we followed up immunosuppressive-naïve active TAK (n = 16) and compared T helper lymphocyte populations and cytokines before and after immunosuppressive therapy. Healthy controls (HC, n = 21) and sarcoidosis (disease control, n = 11) were compared against TAK.ResultsTAK had higher Th17, Th17.1 and PD1+Th17 lymphocytes than HC (p < 0.001), and higher PD1+CD4+ T lymphocytes than sarcoidosis (p < 0.001). Th17 lymphocytes associated with active TAK after multivariable-adjusted logistic regression (p = 0.008). TAK had greater cytokine secretion from PBMCs (IFN-γ, IL-17A, IL-10 versus HC; IL-6, TNF-α, IL-1β versus HC or sarcoidosis) (p < 0.05). In-vitro, PBMCs from TAK showed reduced secretion of all inflammatory cytokines with tacrolimus, with synergistic reduction in IL-17A, IL-6, IL-1β and IL-10 following addition of tadalafil to tacrolimus. Serial follow-up of immunosuppressive-naïve TAK (n = 16) showed reduction in serum IL-6 and TGF-β1 (p < 0.05) and IL-6 in culture supernatant (p < 0.05) following immunosuppressive therapy.ConclusionNovel Th17 sub-populations (Th17.1 and PD1+Th17) are elevated in TAK. Th17 lymphocytes associate with active TAK. In-vitro experiments on cultured PBMCs suggest promise for further evaluation of a combination of immunosuppressive tacrolimus with anti-fibrotic tadalafil (or other anti-fibrotic therapies) in clinical trials of TAK.

Inhibition of IL17A Using an Affibody Molecule Attenuates Inflammation in ApoE-Deficient Mice.

The balance between pro- and anti-inflammatory cytokines released by immune and non-immune cells plays a decisive role in the progression of atherosclerosis. Interleukin (IL)-17A has been shown to accelerate atherosclerosis. In this study, we investigated the effect on pro-inflammatory mediators and atherosclerosis development of an Affibody molecule that targets IL17A. Affibody molecule neutralizing IL17A, or sham were administered in vitro to human aortic smooth muscle cells (HAoSMCs) and murine NIH/3T3 fibroblasts and in vivo to atherosclerosis-prone, hyperlipidaemic ApoE−/− mice. Levels of mediators of inflammation and development of atherosclerosis were compared between treatments. Exposure of human smooth muscle cells and murine NIH/3T3 fibroblasts in vitro to αIL-17A Affibody molecule markedly reduced IL6 and CXCL1 release in supernatants compared with sham exposure. Treatment of ApoE−/− mice with αIL-17A Affibody molecule significantly reduced plasma protein levels of CXCL1, CCL2, CCL3, HGF, PDGFB, MAP2K6, QDPR, and splenocyte mRNA levels of Ccxl1, Il6, and Ccl20 compared with sham exposure. There was no significant difference in atherosclerosis burden between the groups. In conclusion, administration of αIL17A Affibody molecule reduced levels of pro-inflammatory mediators and attenuated inflammation in ApoE−/− mice.

P085 Paneth cells translate dietary lipid exposure into gut inflammation

Abstract Background Dietary constituents impact the development and course of IBD (1). Especially a Western style diet is associated with an increased incidence and prevalence of IBD (2). The role of polyunsaturated fatty acids (PUFA) in the development of IBD is broadly discussed. Recently we could show that PUFAs elicit a pro-inflammatory response in intestinal epithelial cells (IEC) in a Glutathione peroxidase 4 (GPX4) deficient setting, which resembled phenotypic hallmarks of human CD (3). GPX4, an anti-oxidative enzyme, was described to be a risk gene for the development of CD (4) and is deficient in IECs derived from inflamed areas of the small intestine of CD patients (3). Stress responses in Paneth cells (PC) trigger small intestinal gut inflammation (5). In this study, we describe PC as originators of GPX4-restricted and PUFA-induced intestinal inflammation. Methods We analyzed PC morphology in mice lacking one allele of GPX4 specifically in IECs (GPX4+/-IEC) by electron microscopy. We generated mice lacking both alleles of Gpx4 specifically in PC (GPX4ΔPC) and exposed these mice (and wildtype controls) to a Western diet enriched with PUFAs for 12 weeks. Intestinal inflammation was assessed by histology and immunohistochemistry. Further we analyzed small intestinal whole tissue cultures and evaluated cytokine production by ELISA. Results Paneth cells of mice lacking one allele of Gpx4 showed morphological signs of ER-stress at baseline and after exposure to a PUFA-WD for 12 weeks when compared to WT mice (Fig 1a). Based on these results, we dissected a role for PC in GPX4-restricted gut inflammation. When fed a PUFA WD for 12 weeks GPX4ΔPC mice developed a CD like enteritis while WT controls did not (Fig 1b). A WD did not induce this inflammatory response in GPX4ΔPC or WT mice. The infiltrating leukocytes contained mainly neutrophils and macrophages (indicated by positive F4/80 staining, Fib 1c). Whole tissue cultures derived from GPX4ΔPC mice fed a PUFA WD produced more Interleukin-6 and CXCL-1, the murine homologue of the human Interleukin-8, compared to whole tissue cultures derived from WT mice fed the same diet (Fig 2). Figure legend 1a) % of PC showing massive, medium or no ER dilatation 1b&amp;c) enteritis score and representative H&amp;E and F4/80 staining 2) IL-6 and CXCL-1 in supernatant of whole tissue jejunal samples Conclusion The presented data identify Paneth cells to be crucially involved in the development of PUFA induced enteritis in genetically susceptible hosts.

Lactiplantibacillus plantarum dfa1 Outperforms Enterococcus faecium dfa1 on Anti-Obesity in High Fat-Induced Obesity Mice Possibly through the Differences in Gut Dysbiosis Attenuation, despite the Similar Anti-Inflammatory Properties.

Fat reduction and anti-inflammation are commonly claimed properties of probiotics. Lactiplantibacillus plantarum and Enterococcus faecium were tested in high fat-induced obesity mice and in vitro experiments. After 16 weeks of probiotics, L. plantarum dfa1 outperforms E. faecium dfa1 on the anti-obesity property as indicated by body weight, regional fat accumulation, serum cholesterol, inflammatory cytokines (in blood and colon tissue), and gut barrier defect (FITC-dextran assay). With fecal microbiome analysis, L. plantarum dfa1 but not E. faecium dfa1 reduced fecal abundance of pathogenic Proteobacteria without an alteration in total Gram-negative bacteria when compared with non-probiotics obese mice. With palmitic acid induction, the condition media from both probiotics similarly attenuated supernatant IL-8, improved enterocyte integrity and down-regulated cholesterol absorption-associated genes in Caco-2 cell (an enterocyte cell line) and reduced supernatant cytokines (TNF-α and IL-6) with normalization of cell energy status (extracellular flux analysis) in bone-marrow-derived macrophages. Due to the anti-inflammatory effect of the condition media of both probiotics on palmitic acid-activated enterocytes was neutralized by amylase, the active anti-inflammatory molecules might, partly, be exopolysaccharides. As L. plantarum dfa1 out-performed E. faecium dfa1 in anti-obesity property, possibly through the reduced fecal Proteobacteria, with a similar anti-inflammatory exopolysaccharide; L. plantarum is a potentially better option for anti-obesity than E. faecium.

Immunomodulatory effects of Inonotus obliquus polysaccharide on splenic lymphocytes infected with Toxoplasma gondii via NF-κB and MAPKs pathways

Context As a medicinal and edible fungus, Inonotus obliquus has been traditionally used to prevent and treat various ailments. Inonotus obliquus polysaccharide (IOP) isolated from I. obliquus processes many biological activities, our series of in vivo studies have shown that IOP protects against Toxoplasma gondii infection. Objective This study aimed to investigate the in vitro immunomodulatory effects and its mechanisms of IOP on mouse splenic lymphocytes infected with T. gondii. Materials and methods Mouse splenic lymphocytes were infected with T. gondii tachyzoites, and treated with different concentrations of IOP. The levels of cytokines and chemokines were measured by enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR). The expression of toll-like receptor 2 (TLR2) and TLR4, and the modulation of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) signaling pathways were determined by Western blot. Results IOP significantly decreased the over-release of cytokine interleukin-1 beta (IL-1β), IL-4, IL-6, interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α) in supernatant from T. gondii-infected splenic lymphocytes. IOP also effectively inhibited the overexpression of cytokines and chemokine macrophage inflammatory protein-1 (MIP-1) and monocyte chemoattractant protein-1 (MCP-1) mRNA. Furthermore, IOP down-regulated TLR2 and TLR4 expressions and inhibited the over-phosphorylation of NF-κB p65 and inhibitor κBα (IκBα) in NF-κB signaling pathway and p38, c-Jun N-terminal kinase (JNK) in MAPKs signaling pathway. By observing the effect of IOP on TNF-α secretion after pretreatment with specific inhibitors, it was further confirmed that IOP was involved in the regulation of NF-κB, p38, and JNK signaling pathways. Conclusions These data indicate that IOP can inhibit the excessive inflammatory response caused by T. gondii infection through modulating NF-κB, p38, and JNK signaling pathways, and thus plays the in vitro anti-T. gondii role.

Effect of the Connexin 43 Coupling to the Biobehavior of Multiple Myeloma Cells

To investigate the effect of gap junction intercellular communication (GJIC) combined by connexin43 (Cx43) and its signal to the biobehavior of multiple myeloma (MM) cells, and its possible mechanism. The mesenchymal stem cell (MSC) cells were isolated and cultured from patients with MM and normal donors. The expression of connexin43 (Cx43) in MSC cells from different sources was detected by RT-PCR and Western blot. The side population (SP) cells were sorted by flow cytometry (FCM). The effect of MSC cells from different sources to the cell cycle, Cx43 expression, colony formation in vitro, stem cell related genes expression, cytokines secretion and chemoresistance in MM SP cells as well as with or without Cx43 inhibitor 18α-glycyrrhetinic acid (18α-GA) was observed. There was no significantly difference between the MSC isolated from normal donor and MM patients. Western blot showed that Cx43 expression in SP cells was up-regulated when the cells were incubated with MSC, and medium containing 18α-GA could partially inhibit it, moreover, it was more significant in MSC cells of MM patients. The ability of colony formation of SP cells in vitro was higher than those of MM cells and MM-MSC could promote the colony formation in a co-culture manner. The effect of MM-MSC to SP cells was down-regulated after 18α-GA was added. RT-PCR showed that there was several important stem cell-related genes including c-myc, Oct-4 Klf-4, and Sox-2 were found in RPMI 8226 cells, but those cells were up-regulated in SP cells (P<0.001). Meanwhile, MM-MSC could up-regulate the expression of c-myc, Klf-4 and Sox-2 (P<0.001), but down-regulate Oct-4 gene in the SP cells. The expression of those genes decreased after 18α-GA was added, but showed no significant difference (P>0.05). Cytometry bead array assays showed that MM-MSCs could secrete high level of IL-6, but the levels of IL-6, IL-10 and TGF-β increased significantly when the MM-MSCs were co-cultured with SP cells (P<0.05), especially the levels of IL-6 and IL-10 were significantly higher than cultured alone. There was no significant change in the levels of bFGF and IL-17 before and after co-cultured. The levels of IL-6, IL-10 and TGF-β in supernatant decreased significantly after GJ inhibitor 18α-GA was added. PI/Annexin V assay showed that MM cells were sensitive to bortezomib (BTZ)-induced apoptosis, but the sensitivity for SP cells was weaker. The ratio of cell apoptosis was 75.2%±0.77% and 8.12%±0.86% (P<0.001), respectively. MM-MSC could down-regulate the cell apoptosis induced by BTZ, while the sensitivity of MM cells to BTZ could be partially recovered after GJ inhibitor was added. MSC derived from MM patients can enhance GJIC to maintain its "hematopoiesis" by up-regulating the expression of Cx43 in MM cells, and at the same time promote cell proliferation and drug recistance by secreting multiple cytokines, which finally contributes to the relapse of MM.

Honokiol alleviates LPS-induced acute lung injury by inhibiting NLRP3 inflammasome-mediated pyroptosis via Nrf2 activation in vitro and in vivo

BackgroundHonokiol (HKL) has been reported to ameliorate lipopolysaccharide (LPS)-induced acute lung injury (ALI). However, its potential mechanism of its protective effects remains unclear. In this study, the protective mechanism of HKL on LPS-induced ALI was explored in vivo and in vitro.MethodsIn vivo, the SD rats were intratracheally instilled with LPS (5 mg/kg) to establish an acute lung injury model and then treated with HKL (1.25/2.5/5 mg/kg) or ML385 (30 mg/kg) intraperitoneally. In vitro, the human bronchial epithelial cell line (BEAS-2B) was stimulated with LPS and ATP to induce pyroptosis and treated with HKL (12.5/25/50 μM). Small interfering RNA (siRNA) technique was used to knockdown Nrf2 in BEAS-2B cells. The protein and mRNA expression levels of Nrf2, HO-1, NLRP3, ASC, CASP1, and GSDMD in cells and lung tissues were detected by western blot and real time-PCR. The expression levels of interleukin (IL)-1β, IL-18, MPO, MDA, and SOD in bronchoalveolar lavage fluid (BALF) and supernatant were determined by ELISA. The degree of pathological injury of lung tissue was evaluated by H&E staining.ResultsThe results showed that HKL could alleviate oxidative stress and inflammatory responses by regulating the levels of MPO, MDA, SOD, IL-1β, IL-18 in supernatant. And it could also inhibit the expression levels of NLRP3, ASC, CASP1, GSDMD via activation of Nrf2 in BEAS-2B cells. Further studies revealed that HKL could attenuate the pathological injury in LPS-induced ALI rats, and the molecular mechanism was consistent with the results in vitro.ConclusionsOur study demonstrated that HKL could alleviate LPS-induced ALI by reducing the oxidative stress and inhibiting NLRP3 inflammasome-mediated pyroptosis, which was partly dependent on the Nrf2 activation.Graphical

Th17/Treg cells regulated by interleukin 6 in the pathogenesis of chronic rhinosinusitis with nasal polyps.

To be investigated whether Th17/Treg cells regulated by Interleukin-6 in the pathogenesis of chronic rhinosinusitis with nasal polyps. The distributions of Th17 and Treg cells in peripheral blood mononuclear cells (PBMCs) and tissues got from 46 CRSwNP patients and 14 controls were evaluated. Th17 and Treg cells and cells-related cytokines in serum were assessed in means of cytometric bead array (CBA) multiplex assays and enzyme-linked immunosorbent assays (ELISAs). Spleen cells were isolated from spleen of 20 normal BALB/c mice (male), isolated and purified with CD4 antibody immunomagnetic bead kit. CD4+ cells were divided into three groups, including TGF-β1, TGF-β1+ IL-6 and control (PBS). Treg and Th17 cells and cells related cytokines were detected by flow cytometry (FCM) after collecting spleen cells. The level of IL-10 and IL-17 in supernatant was measured by ELISA. Th17/Treg ratio and the level of IL-6 in both ECRSwNP (P < 0.05) and non-ECRSwNP (P < 0.05) were significantly increased when compared with control group, these were consistent with the previous findings. Experiments in vitro suggested that the level of Th17 cells in IL-6+ TGF-β1 group was significantly increased than TGF-β1 group and control group (P < 0.05). The ratio of cells expressed RoRγt in IL-6+ TGF-β1 group was much higher than TGF-β1 group (P < 0.05). IL-6 might regulate the function of Th17 and Treg cells and the Th17/Treg ratio and have a role in the pathogenesis of CRSwNP.