IL-10 Protein Research Articles

Piperine Derived from Piper nigrum L. Inhibits LPS-Induced Inflammatory through the MAPK and NF-κB Signalling Pathways in RAW264.7 Cells.

Piperine, an important natural product, has a good anti-inflammatory effect. However, few researchers have studied its mechanism in these pathways. The objective of this research was to evaluate the molecular mechanism underlying the anti-inflammatory responses of piperine in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. The purification and characterization of piperine from Piper nigrum L. were determined by HPLC, UPLC-Q-TOF-MS and 1H NMR. Then, the anti-inflammatory activity was evaluated by a reagent test kit, ELISA kits, RT-PCR and Western blot experiments. The results suggested that piperine (90.65 ± 0.46% purity) at a concentration of 10–20 mg/L attenuated the production of NO and ROS, downregulated the protein and mRNA expression levels of TNF-α, IL-1β and IL-6, and upregulated the protein and mRNA transcription levels of IL-10. Meanwhile, the Western blot results indicated that piperine could inhibit the phosphorylation levels of the ERK, JNK, p38 and p65 proteins. Our findings suggest that piperine is a potential anti-inflammatory substance, whose molecular mechanism may be to regulate the key factors of the NF-κB and MAPK signalling pathways.

Effect of acupuncture preconditioning combined with PI3K blocker LY294002 on airway inflammation in asthmatic rats

To observe the effect of acupuncture preconditioning combined with PI3K blocker LY294002 on the expression of PI3K and Akt proteins and genes in the lung tissue and the contents of serum IL-12 and IL-13 in asthmatic rats, so as to explore its preprotective mechanism underlying improving asthma. Sixty male Wistar rats were randomly divided into blank control, model, acupuncture pretreatment + blank, acupuncture pretreatment, acupuncture pretreatment + LY294002 and LY294002 groups (n=10 in each group). The asthma model was established by intraperitoneal injection of mixture solution of OVA and Al(OH)3 and followed inhalation of 1%OVA for 30 min, once daily for 7 days. Rats of the blocker groups received inhalation of atomized LY294002 solution for 30 min before inhalation of 1% OVA, and acupuncture was applied to "Feishu"(BL13), "Dazhui"(GV14) and "Fengmen"(BL12) for 20 min, once daily for 7 days before modeling. H.E. staining was used to assess histopathological changes of the lung tissue, and ELISA was used to detect the contents of serum IL-12 and IL-13. The immunoactivity of PI3K and Akt and expression of Akt mRNA of the lung tissue were detected by using immunohistochemistry and fluorescence quantitative real-time PCR, separately. Compared with the blank control group, the content of serum IL-12 was significantly decreased (P<0.01), and the content of serum IL-13, the expression levels of PI3K, Akt protein and Akt mRNA were remarkably increased (P<0.01) in the model group. In comparison with the model group, the content of serum IL-12 in the pretreatment, pretreatment + LY294002 and LY294002 groups was significantly increased (P<0.01, P<0.05), while the content of IL-13 and the expression levels of PI3K, Akt protein and Akt mRNA were considerably decreased (P<0.01, P<0.05) in the acupuncture pretreatment, acupuncture pretreatment+LY294002 and LY294002 groups. The therapeutic effect of acupuncture pretreatment+LY294002 was obviously superior to that of simple acupuncture pretreatment and LY294002 (except PI3K and Akt in the LY294002 group) in up-regulating serum IL-12 level, and in down-regulating serum IL-13, and PI3K and Akt protein levels in the lung tissue (P<0.01). H.E. staining showed severe inflammatory factor infiltration in the bronchus and pulmonary interstitium, and obvious bronchial lumen narrowing with increased exudate in rats of the model group, which was relatively milder in rats of the acupuncture pretreatment, acupuncture pretreatment+LY294002 and LY294002 groups. There were no significant diffe-rences between blank control and pretreatment+blank groups in all of the above indicators （P>0.05）. Acupuncture preconditioning can inhibit airway inflammation in asthmatic rats, which may be associated with its functions in down-regulating the levels of pulmonary PI3K and Akt and serum IL-13 and up-regulating the content of serum IL-12. Acupuncture preconditioning combined with LY294002 has the best effect.

Ethanol Extracts of Rice Bran and Whole Grain Adlay Seeds Mitigate Colonic Inflammation and Damage in Mice with Colitis.

Ulcerative colitis (UC) is a chronic inflammatory bowel disease with frequent relapsing inflammation in the colon. Whole grains have been promoted as healthy and sustainable foods; however, the use of whole gains in UC is inconclusive. The aim of this study was to investigate the effects of ethanol extracts of rice bran (RBE) and whole-grain adlay seeds (ADE) on inflammation, oxidative stress, and colonic damage in UC. Male C57BL/6JNarl mice were intra-rectal injected twice with 2,4-dinitrobenzene sulfonic acid to induce (day 0) and reactivate (day 21) UC. Control mice were fed AIN-93M diet (R group) and injected with a vehicle. UC mice were fed AIN-93M diet (UC group) supplemented with RBE (RBE group) or ADE (ADE group) for 21 days. The results showed that the UC group had an increased disease activity index, plasma interleukin (IL)-6 and glutathione levels, microscopic injury scores, and inflammatory cytokine and chemokine levels in the colon and decreased colonic claudin-4 compared to the R group. RBE and ADE supplementation significantly reduced UC-elevated plasma IL-6 and colonic glutathione and pro-inflammatory cytokines and a chemokine. In addition, RBE and ADE supplementation significantly decreased T-helper-cell-associated cytokines in the plasma and colon. Moreover, RBE supplementation increased colonic IL-10 and tight junction protein claudin-4 levels, and ADE supplementation alleviated diarrhea in UC mice. In conclusion, these results suggest that RBE and ADE may mitigate colonic inflammation, oxidative stress, and damage in UC relapse.

ADAM-10 Regulates MMP-12 during Lipopolysaccharide-Induced Inflammatory Response in Macrophages.

A disintegrin and metalloprotease 10 (ADAM-10), a member of the ADAM protease family, has biological activities related to TNF-α activation, cell adhesion, and migration, among other functions. Macrophages are important immune cells that are involved in the inflammatory response of the body. ADAM-10 is involved in inflammatory responses, but the specific regulatory mechanisms are not fully understood. In this study, we investigated the regulatory mechanism of ADAM-10 in the lipopolysaccharide-promoted proliferation (LPS) of the macrophage inflammatory response. Differentially expressed or regulated proteins were identified in interfered ADAM-10 (sh ADAM-10) macrophages using tandem mass tag (TMT) proteomics. The changes and regulatory role of ADAM-10 during LPS-induced inflammatory response in normal, interfering, and overexpressing ADAM-10 (EX ADAM-10) cells were determined. Results indicated that ADAM-10 interference affected inflammation-related pathways and reduced matrix metalloproteinase 12 (MMP-12) protein levels, as identified by TMT proteomics. In normal cells, LPS decreased ADAM-10 gene expression, but promoted ADAM-10 secretion, MMP-12 and TNF-α gene expression, and MMP-12, iNOS, IL-10, and cyclinD1 protein expression. Additionally, ADAM-10 knockdown decreased macrophage viability in sh-ADAM-10 cells. Moreover, an MMP-12 inhibitor had no impact on the viability effect of LPS on cells or the expression of ADAM-10. iNOS expression decreased, whereas IL-10 expression increased after ADAM-10 depletion. ADAM-10 knockdown decreased MMP-12, iNOS, TNF-α, IL-1β, and FKN, while overexpression had an opposite effect. ADAM-10 overexpression further increased MMP-12, iNOS, and TNF-α gene expression in response to LPS. Cell viability was increased in EX ADAM-10 cells, and ADAM-10 secretion was further increased in the EX and LPS groups. Flow cytometry and immunofluorescence staining revealed that EX-ADAM 10 cells had increased iNOS expression, which acted as an IL-6 expression driver. In summary, we found that ADAM-10 is activated by LPS and positively participates in LPS-stimulated macrophage inflammatory responses by positively regulating MMP-12 during the inflammatory process.

Increased seizure sensitivity in pregnant mice with genetic knockdown of acid sensing ion channel 2a is associated with impaired hippocampal inflammatory response.

Acid sensing ion channels (ASICs) are mechano- and chemo-receptor channels that are activated by drops in extracellular pH as occurs after neurotransmission. In our previous study, we demonstrated that mice subjected to reduced utero-placental perfusion pressure during pregnancy, to mimic the pregnancy complication of preeclampsia, have reduced hippocampal expression of ASIC2a protein. We also showed that pregnant mice with heterozygous expression of ASIC2a (+/-) had increased sensitivity and severity to pentylenetetrazol-induced seizures; however, the mechanisms by which this occurs remain unclear. The purpose of this study was to investigate key molecular targets involving neurotransmission and inflammation that are differentially changed following seizure exposure in pregnant ASIC2a +/- mice. On gestational day 18.5, ASIC2a wild-type (+/+, n = 7) and +/- (n = 14) mice were injected with 40 mg/kg pentylenetetrazol and monitored for 30 min. Western blot and ELISA analysis revealed no difference in hippocampal synaptosome glutamate-related proteins but an increase in GABA concentration in pregnant +/- mice. Using ELISA and multiplex assays, we found a significant decrease in serum TNFα, and a decreased concentration of pro-inflammatory cytokines and chemokines in hippocampal cytosolic fraction. Significant reductions in IL-1β, IL-3, IL-12 (p70), eotaxin, interferon gamma, and macrophage inflammatory protein (MIP-1β), in the hippocampal cytosolic fractions of +/- mice were observed compared to +/+ mice. Additionally, there was no difference in hippocampal microglia density or activation in pregnant ASIC2a+/+ vs. +/- mice. These results support the hypothesis that pregnant mice with reduced ASIC2a may not be able to mount an inflammatory response following acute seizure exposure.

Human differentiated eosinophils release IL-13 in response to IL-33 stimulation.

ObjectiveEosinophils are hallmarks in allergic type 2 inflammation and are known to release cytotoxic granule proteins that contribute to inflammation. Eosinophils develop in the bone marrow from hematopoietic stem cells and once mature, have a limited lifespan in culture, making them difficult to study ex vivo. IL-33 has increasingly been shown as a key regulator of type 2 inflammation via signaling through its receptor, ST2. The present study was conducted to detail a method of eosinophil differentiation from hematopoietic stem cells and determine the response to IL-33.MethodsCD34+ and CD14+ cells were isolated from donor apheresis cones and differentiated into eosinophils or macrophage controls, respectively. Morphologic, transcriptional and protein analyses were performed to validate this method of eosinophil differentiation. The effect of IL-33 on differentiated eosinophils was assessed using qPCR, immunofluorescence, and multiplex cytokine array.ResultsCD34 differentiated eosinophils appear morphologically similar by H&E and express eosinophil peroxidase (EPX) protein as well as the conventional eosinophil transcripts EPX, CLC, and MBP. In addition, differentiated eosinophils expressed both isoforms of the IL-33 receptor, ST2L and sST2 throughout the differentiation process. Transcript levels of both IL-33 receptors were up-regulated by treatment with IL-33 at earlier timepoints in the differentiation. These cells also expressed IL-4 and IL-13 mRNA which were up-regulated by IL-33 as well. Notably, IL-13 expression was significantly higher with IL-33 treatment compared to media control at every timepoint measured. IL-33 significantly increased cellular secretion of IL-13 protein at most timepoints throughout differentiation. IL-8, LIF, CCL1, CCL5, CCL7, and CCL8 were also significantly secreted after IL-33 stimulation.ConclusionsOur findings suggest that CD34 differentiated eosinophils are morphologically and phenotypically similar to peripheral eosinophils. The release of specific cytokines in direct response to IL-33 may contribute to the pathogenesis of type 2 inflammation and facilitates new avenues for studying eosinophils as effector cells in vitro.

PI3K/AKT/mTOR Signaling Pathway Is Downregulated by Runzaoling (RZL) in Sjögren's Syndrome.

Infiltration and aggregation of lymphocytes in exocrine glands are the basic pathological manifestations of Sjögren's syndrome (SS), and the incidence of SS has been increasing year by year in recent years. To explore the potential signaling pathway of Runzaoling (RZL) in alleviating SS, the possible targets of RZL in SS were firstly explored through network pharmacology, and then, the regulation of PI3K/AKT/mTOR signaling in NOD mice and Th17 cells was verified. 75 8-week-old NOD mice were casually classified into 5 groups: model; hydroxychloroquine; high, medium, and low dose RZL groups, with 15 in each; and 15 BALB/c mice were employed as control group. After 10 weeks of continuous intragastric administration in mice and 24 hours of drugs intervention in Th17 cells, histopathology was observed by HE staining, and the gene transcription levels were identified by real-time quantitative PCR (RT-qPCR). The protein expressions were detected by western blotting (WB). The findings showed that high and medium dose RZL group could attenuate the submandibular gland tissue damage. The results indicated that the mRNA expressions of PI3K, AKT, mTOR, STAT3, and IL-17 in SS mice and in IL-17 stimulation of Th17 cells were dramatically increased compared with control group and decreased to varying degrees after RZL intervention. The trend of phosphorylated PI3K/AKT/mTOR and STAT3 and IL-17 protein expression in NOD mice and Th17 cells were consistent with mRNA. RZL can downregulate STAT3 and IL-17 expressions in the submandibular gland of NOD mice and in Th17 cells via regulating the PI3K/AKT/mTOR signaling pathway. Moreover, RZL could reduce the activation of CD4+ T lymphocyte differentiation to Th17 cells.

IL-17A in Human Liver: Significant Source of Inflammation and Trigger of Liver Fibrosis Initiation.

IL-17A is considered to guide liver inflammation and fibrosis. From twenty-two human liver samples of different fibrosis stages (F0 to F4), IL-17A, IL-22, and TGFβ1 protein expression in liver tissue lysates were analyzed. Ten paired samples of liver tissue (F0–F1 stage) and blood from the same patient were used to analyze intrahepatic and blood T-lymphoid IL-17A+ cells by flow cytometry. The analyses have been performed regardless of pathology, considering the stage of fibrosis. Human liver tissue was used for the primary human liver slice cultures, followed by subsequent cytokine stimulation and fibrotic markers’ analysis by ELISA. IL-17A production in human liver tissue was significantly higher in the early fibrotic stage compared with the advanced stage. Th17 T cells and, to a lesser extent, MAIT cells were the main sources of IL-17A in both compartments, the liver and the blood. Moreover, the presence of liver Th17IL-17A+INFγ+ cells was detected in the liver. IL-17A stimulation of human liver slice culture increased the expression of profibrotic and pro-inflammatory markers. IL-17A, secreted by Th17 and MAIT cells in the liver, triggered fibrosis by inducing the expression of IL-6 and profibrotic markers and could be a target for antifibrotic treatment. Further amplitude studies are needed to confirm the current results.

Glycyrrhizic Acid Protects Experimental Sepsis Rats against Acute Lung Injury and Inflammation.

Background The incidence of acute lung injury/acute respiratory distress (ALI/ARDS) is high in sepsis aggravating morbidity and mortality. Glycyrrhizic acid (GA) has pharmacological activities in the treatment of inflammation and antiviral. Materials and Methods Sepsis rats were constructed by the cecal ligation and puncture (CLP) surgery. After GA (25 and 50 mg/kg) injection, the survival rate, blood oxygen, biochemical indexes, myeloperoxidase (MPO) activity, and wet/dry weight ratio of the lung were observed. The bronchoalveolar lavage fluid was collected to count the cells and measure the level of TNF-α, IL-1β, IL-10, and high mobility group box-1 protein (HMGB1). Lung tissue sections were taken to observe the levels of histopathological injury and apoptosis by HE and TUNEL staining. The levels of HMGB1, TLR4, p-38 MAPK, NF-κB, and ERK1/2 proteins were observed by immunohistochemistry and Western blot. Results GA treatment improved the survival rate, blood oxygen, ALT, AST, BUN, and Scr of CLP rats. It could advance the MPO activity, the wet/dry weight ratio, histopathological injury, apoptosis, and the IL-10 level in the lung. After GA injection, the number of total cells, neutrophils, and macrophages in the CLP rats was reduced and the levels of TNF-α, IL-1β, HMGB1, TLR4, p-38 MAPK, and ERK1/2 in the CLP rat were also repressed. Conclusions GA treatment may improve the sepsis-induced ALI/ARDS and inflammation by inhibiting HMBG1. This study provided an experimental basis for the prevention and treatment of ALI/ARDS caused by sepsis.

Effects of a whole food diet on immune function and inflammatory phenotype in healthy dogs: A randomized, open-labeled, cross-over clinical trial.

Whole foods in humans decrease inflammation and risk for various diseases, as well as increase weight loss and immune function. Nutrition has been shown to be an integral component in the management of various diseases in dogs but the immunologic and anti-inflammatory effects of whole food diets have not been explored. Therefore, our objective was to assess the effect of feeding a whole food diet on immune function and inflammatory phenotype in healthy dogs. A prospective, randomized, open-labeled, cross-over clinical trial was performed. Sixteen healthy client-owned dogs were fed either a whole food or an extruded dry diet, and after 67 days, they were fed the alternate diet for an additional 67 days. Blood samples were obtained at the completion of each treatment arm (i.e., days 67 and 134). Serum c-reactive protein (CRP), haptoglobin (Hp), and serum amyloid-A (SAA) were measured with ELISA assays. Whole blood cultures were performed with exposure to a phosphate-buffered solution (PBS), lipopolysaccharide (LPS), and lipoteichoic acid (LTA). A canine specific multiplex bead-based assay was then used to measure tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-10, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-2, IL-8, and monocyte chemoattractant protein (MCP)-1 concentrations. Granulocyte/monocyte (GM) phagocytosis and oxidative burst associated with Escherichia coli were evaluated via flow cytometry. Dogs fed a whole food diet had significantly lower TNF-α-to-IL-10 ratios (P = 0.05) and higher production of IL-8 (P = 0.03) with LTA-exposed leukocytes compared to dogs fed an extruded dry diet. There were no between-treatment differences in the remaining leukocyte cytokine responses, serum CRP, Hp, SAA concentrations, or GM phagocytic and oxidative burst capacities. Whole food diets could have immunomodulatory effects in dogs. Future studies in non-healthy dogs are warranted.

MicroRNA-155 acts as an anti-inflammatory factor in orbital fibroblasts from Graves' orbitopathy by repressing interleukin-2-inducible T-cell kinase.

To investigate the role of microRNA (miR)-155 in inflammation in an in-vitro model of Graves’ orbitopathy (GO). The expression levels of miR-155 were compared between GO and non-GO orbital tissues. The effects of inflammatory stimulation of interleukin (IL)-1β and tumour necrosis factor alpha (TNF-α) on miR-155 expression on GO and non-GO orbital fibroblasts (OFs) were investigated. The effects of miR-155 mimics and inhibitors of inflammatory proteins and IL-2-inducible T-cell kinase (ITK) expression were examined, along with those related to the knockdown of ITK with siITK transfection on inflammatory proteins. We also examined how ITK inhibitors affect miR-155 expression in GO and non-GO OFs. The expression levels of miR-155 were higher in GO orbital tissues than in non-GO tissue. The overexpression of miR-155 was induced by IL-1β and TNF-α in OFs from GO and non-GO patients. IL-1β-induced IL-6 (ICAM1) protein production was significantly reduced (increased) by miR-155 mimics and inhibitors. The mRNA and protein levels of ITK were downregulated by overexpressed miR-155 via miR-155 mimics. Knockdown of ITK via siITK transfection induced a decrease in the expression levels of ITK, IL-17, IL-6, IL-1β, and TNF-α protein. The expression of miR-155 was significantly downregulated by treatment with ITK inhibitors and Bruton’s tyrosine kinase (BTK)/ITK dual inhibitors in a time-dependent manner. Our results indicated a potential relationship between miR-155 and ITK in the context of GO OFs. The overexpression of miR-155 repressed ITK expression and relieved inflammation. Thus, miR-155 appears to have anti-inflammatory effects in GO OFs. This discovery provides a new concept for developing GO treatment therapeutics.

The radiographic assessment of lung edema score of lung edema severity correlates with inflammatory parameters in patients with coronavirus disease 2019-Potential new admission biomarkers to predict coronavirus disease 2019 worsening.

BackgroundCoronavirus disease 2019 (COVID-19) has placed enormous pressure on intensive care units (ICUs) and on healthcare systems in general. A deeper understanding of the pathophysiology of the most severe forms of COVID-19 would help guide the development of more effective interventions. Herein, we characterized the inflammatory state of patients with COVID-19 of varying degrees of severity to identify admission biomarkers for predicting COVID-19 worsening.DesignAdmission blood samples were obtained from 78 patients with COVID-19. Radiographic assessment of lung edema (RALE) scoring was calculated by imaging. Platelet and leukocyte counts were measured by flow cytometry, and plasma levels of C-reactive protein were assessed by immunoturbidimetry, and interleukin (IL)-8/CXCL8, IL-10, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and monocyte chemoattractant protein-1 (MCP-1/CCL2) levels by enzyme-linked immunosorbent assay (ELISA).ResultsThe RALE score correlated with several admission hemogram (platelets, neutrophils, and lymphocytes) and inflammatory (IL-8/CXCL8, MCP-1/CCL2, IL-10, and C-reactive protein) parameters. COVID-19 worsening, based on the need for oxygen (Δoxygen supply) during hospitalization, correlated negatively with admission lymphocyte counts but positively with neutrophil-to-lymphocyte ratio and with plasma levels of the inflammatory parameters correlating with RALE score.ConclusionOur data indicate a correlation between the RALE score and Δoxygen supply and admission inflammatory status. The identification of a panel of biomarkers that reflect COVID severity might be useful to predict disease worsening during hospitalization and to guide clinical management of COVID-19-related complications. Finally, therapies targeting IL-8/CXCL8- or IL-10 activity may offer therapeutic approaches in COVID-19 treatment.

Detergent exposure induces epithelial barrier dysfunction and eosinophilic inflammation in the esophagus.

Eosinophilic esophagitis (EoE) is a chronic allergic disease associated with type 2 inflammation and epithelial barrier dysfunction. The etiology is unknown, however, genetic heritability studies suggest environmental factors play a key role in pathogenesis. Detergents, such as sodium dodecyl sulfate (SDS), are common ingredients in household products such as dish soap and toothpaste. We hypothesized detergent exposure decreases epithelial barrier function and induces esophageal inflammation. Immortalized esophageal epithelial cells (EPC2) were cultured in air-liquid interface (ALI) and exposed to SDS. Barrier function/activity was assessed by transepithelial electrical resistance (TEER), FITC-dextran flux, and RT-PCR. Additionally, SDS-treated mouse esophageal organoids were evaluated for morphology. To investigate the effects of SDS in vivo, mice were treated with 0.5% SDS in drinking water for 14 days. Esophagi were assessed by gross morphology, histopathology, protein expression, and bulk RNA sequencing. When EPC2 cells were exposed to SDS (5μg/ml) for 96 h, TEER decreased (p=0.03), and FITC-dextran flux increased (p=0.0002). mRNA expression of IL-33 increased 4.5-fold (p=0.02) at 6h and DSG1 decreased (p < 0.0001) by 72 h. Disrupted epithelial integrity was noted in SDS-treated esophageal organoids. When mice were exposed to SDS, they showed increased esophageal width, chemokine, and metalloprotease levels. Mice treated with SDS also showed increased IL-33 protein expression, basal zone hyperplasia, CD4+ cell infiltration, and esophageal eosinophilia. RNA sequencing revealed upregulation of immune response pathway genes. Exposure to SDS decreases esophageal barrier integrity, stimulates IL-33 production, and promotes epithelial hyperplasia and tissue eosinophilia. Detergents may be a key environmental trigger in EoE pathogenesis.

Pseudomonas Aeruginosa Lung Infection Subverts Lymphocytic Responses through IL-23 and IL-22 Post-Transcriptional Regulation.

Pseudomonas aeruginosa (P.a) is a pathogen causing significant morbidity and mortality, particularly in hospital patients undergoing ventilation and in individuals with cystic fibrosis. Although we and others have investigated mechanisms used by P.a to subvert innate immunity, relatively less is known about the potential strategies used by this bacterium to fight the adaptive immune system and, in particular, T cells. Here, using RAG KO (devoid of ‘classical’ αβ and γδ TCR T lymphocytes) and double RAG γC KO mice (devoid of T, NK and ILC cells), we demonstrate that the lymphocytic compartment is important to combat P.a (PAO1 strain). Indeed, we show that PAO1 load was increased in double RAG γC KO mice. In addition, we show that PAO1 down-regulates IL-23 and IL-22 protein accumulation in the lungs of infected mice while up-regulating their RNA production, thereby pointing towards a specific post-transcriptional regulatory mechanism not affecting other inflammatory mediators. Finally, we demonstrate that an adenovirus-mediated over-expression of IL-1, IL-23 and IL-7 induced lung neutrophil and lymphocytic influx and rescued mice against P.a-induced lethality in all WT, RAG γC KO and RAG γC KO RAG-deficient mice, suggesting that this regimen might be of value in ‘locally immunosuppressed’ individuals such as cystic fibrosis patients.

Bacillus coagulans in Combination with Chitooligosaccharides Regulates Gut Microbiota and Ameliorates the DSS-Induced Colitis in Mice.

ABSTRACTUlcerative colitis (UC) are chronic inflammatory disorders, which may be caused by intestinal barrier dysfunction, immune system disorders and intestinal microbiota dysbiosis. Synbiotic, the combination of probiotics and prebiotics, is thought to be a pragmatic approach in mitigating inflammation in UC. Bacillus coagulans has been recognized as a potential probiotic for treating intestinal diseases because of its favorable industrial and probiotic properties, including sporulation and lactic acid production. In this study, we evaluated the treatment effects of the B. coagulans FCYS01 spores with or without the chitooligosaccharides (COSs) on UC generated using dextran sulfate sodium (DSS) in mice. Supplementation of B. coagulans spores, prebiotic COSs or the synbiotic (the spores + COSs) had a significant positive effect on DSS-induced UC. The disease activity index and histological damage score were significantly reduced after these supplementations. Compared to DSS group, these supplementations also significantly modulated the cytokines IL-4, IL-6, IL-8, IL-10, and C-reactive protein (CRP) levels and significantly maintained expressions of tight junction proteins and mucin protein and promotes recovery of the intestinal barrier. In addition, these supplementations regulate the composition of gut microbiota and improve the production of short-chain fatty acids (SCFAs), through enrichment of SCFA-producing bacteria, such as Akkermansia and Ruminococcus species. In summary, the synbiotic ameliorated the overall inflammatory status of the experimental UC model and showed a better treatment effect than B. coagulans or COSs did alone as revealed by the markers such as, colon length, IL-4 and Occludin levels.IMPORTANCE Probiotic and prebiotic are believed to be useful in alleviating the inflammatory, thereby resolving or preventing the severity of UC. Spore-forming bacteria Bacillus coagulans show advantages of stability and probiotic effects, being suggested as the important probiotics for UC treatment. Here, we demonstrate that administration of B. coagulans spores, chitooligosaccharides (COSs), or the synbiotic attenuates DSS-induced colitis and significantly correlates with altered gut immune responses. The treatment effect of the synbiotic is inferred to be relied on the enrichment of probiotic bacteria, such as Akkermansia and Ruminococcaceae species, which are reported to be crucial important for gut health. Our findings facilitate the development of therapeutic and preventive strategies for UC using spore-forming lactic acid bacteria in combination with COSs.

Glaesserella parasuis induces IL-17 production might through PKC-ERK/MAPK and IκB/NF-κB signaling pathways

In our previous study, the outer membrane protein P2 of Glaesserella parasuis (G. parasuis) induced the production of interleukin-17A (IL-17A) in host cells. However, how the G. parasuis infection produces IL-17A in the host is unknown. In this study, at 48 hpi in piglets, the proportion of Th17 cells in the peripheral blood significantly increased as determined by flow cytometry, but the overall proportion was low. IL-17 mRNA and protein levels significantly increased in the lungs and spleens of infected piglets. Histopathological examination staining, immunohistochemistry and viability counts, revealed that IL-17 expression might be positively correlated with bacterial content and the degree of pathological injury in lung and spleen tissues. Furthermore, when infected with the G. parasuis Nagasaki strain compared with avirulent strain C5, the primary porcine alveolar macrophages (PAMs) significantly produced IL-17 in a time-dose dependent manner. In addition, we demonstrated that PKC, MEK and NF-κB signaling pathways were essential for G. parasuis-induced IL-17 production because the addition of corresponding inhibitors dramatically reduced IL-17 mRNA production. The phosphorylation levels of PKC, ERK1/2 and IκB in G. parasuis-infected primary PAMs also increased significantly as the infection dose increased as assessed by western blotting. Thus, these finding imply that IL-17 induced by G. parasuis infection is dependent on activation of PKC-ERK/MAPK and IκB/NF-κB signaling pathways.

Immune Transcriptome and Secretome Differ between Human CD71+ Erythroid Cells from Adult Bone Marrow and Fetal Liver Parenchyma.

CD71+ erythroid cells (CECs) were only known as erythrocyte progenitors not so long ago. In present times, however, they have been shown to be active players in immune regulation, especially in immunosuppression by the means of ROS, arginase-1 and arginase-2 production. Here, we uncover organ-of-origin differences in cytokine gene expression using NanoString and protein production using Bio-Plex between CECs from healthy human adult bone marrow and from human fetal liver parenchyma. Namely, healthy human adult bone marrow CECs both expressed and produced IFN-a, IL-1b, IL-8, IL-18 and MIF mRNA and protein, while human fetal liver parenchyma CECs expressed and produced IFN-a, IL15, IL18 and TNF-b mRNA and protein. We also detected TLR2 and TLR9 gene expression in both varieties of CECs and TLR1 and NOD2 gene expression in human fetal liver parenchyma CECs only. These observations suggest that there might be undiscovered roles in immune response for CECs.

Changes in Th9 and Th17 lymphocytes and functional cytokines and their relationship with thyroid-stimulating hormone receptor antibodies at different stages of graves' disease.

ObjectiveGraves’ disease (GD) is an organ-specific autoimmune disease characterized by the production of thyroid-stimulating antibodies (TSAb). The newly discovered CD4+ T helper cells, Th9 and Th17 lymphocytes, have been confirmed to be closely associated with a variety of immune diseases. However, relationships with the onset and development of GD remain unclear. The purpose of this study was to investigate the roles of Th9 and Th17 in the pathogenesis and prognosis of GD.PatientsWe recruited 26 patients with newly diagnosed GD, 45 patients with GD in remission, and 20 healthy individuals.MeasurementsThyroid function and autoantibodies were evaluated using chemiluminescence immunoassays. Th9 and Th17 cells were analyzed using flow cytometry. The expression of Foxo1, IRF-4, RORc, IL-9, and IL-17 mRNA was examined using real-time PCR, and IL-9 and IL-17 protein levels were measured using enzyme-linked immunosorbent assay.ResultsTh9, Th17, and characteristic cytokines IL-9 and IL-17 in the GD-untreated group were significantly higher than those in the control and remission groups. The above indexes significantly decreased in the remission group, with the levels in the TRAb− remission group being similar to those in the normal group, while in the TRAb+ remission group, levels were differentially increased. TRAb titer was positively correlated with the levels of Th9, Th17, and their functional cytokines.ConclusionsTh9 and Th17 cells may be involved in the pathogenesis and disease outcome of GD, which could provide a new direction for developing immunotherapy for patients with GD.

Glycosaminoglycan scaffolding and neural progenitor cell transplantation promotes regenerative immunomodulation in the mouse ischemic brain

Ischemic stroke is a leading cause of morbidity and mortality, with limited treatments that can facilitate brain regeneration. Neural progenitor cells (NPCs) hold promise for replacing tissue lost to stroke, and biomaterial approaches may improve their efficacy to overcome hurdles in clinical translation. The immune response and its role in stroke pathogenesis and regeneration may interplay with critical mechanisms of stem cell and biomaterial therapies. Cellular therapy can modulate the immune response to reduce toxic neuroinflammation early after ischemia. However, few studies have attempted to harness the regenerative effects of neuroinflammation to augment recovery. Our previous studies demonstrated that intracerebrally transplanted NPCs encapsulated in a chondroitin sulfate-A hydrogel (CS-A + NPCs) can improve vascular regeneration after stroke. In this paper, we found that CS-A + NPCs affect the microglia/macrophage response to promote a regenerative phenotype following stroke in mice. Following transplantation, PPARγ-expressing microglia/macrophages, and MCP-1 and IL-10 protein levels are enhanced. Secreted immunomodulatory factor expression of other factors was altered compared to NPC transplantation alone. Post-stroke depression-like behavior was reduced following cellular and material transplantation. Furthermore, we showed in cultures that microglia/macrophages encapsulated in CS-A had increased expression of angiogenic and arteriogenic mediators. Neutralization with anti-IL-10 antibody negated these effects in vitro. Cumulatively, this work provides a framework for understanding the mechanisms by which immunomodulatory biomaterials can enhance the regenerative effects of cellular therapy for ischemic stroke and other brain injuries.

Interleukin-10 plasmid delivery by polymeric nanocarrier shows efficient and safe tissue repair in acute muscle damage models in mice

We established a custom-designed nanocarrier for in vivo delivery of an immunomodulatory plasmid capable of facilitating tissue repair in acute muscle damage models in mice. The nanocarrier was constructed by selecting from versatile cationic designs that were used to complex with a negatively charged plasmid, while the excessive positive charge was shielded by the poly (glutamic acid)-polyethylene glycol co-polymer. The best-performing formulation was identified by high throughput screening in macrophages, in which we obtained the hierarchical ranking in terms of reporter gene expression (GFP plasmid, pGFP) and macrophage polarization status (interleukin 10 plasmid, pIL-10). For proof-of-principle, we performed intramuscular (IM) injection of pGFP nanocarrier in a cardiotoxin-induced muscle injury model. After confirming the in vivo GFP transfection, we demonstrated protected delivery of pIL-10 to the injured muscle site, leading to an accelerated tissue repair outcome. Immunophenotyping analysis revealed the increased number of M2 macrophages and regulatory T cells, as well as increased IL-10 production at muscle site. The effectiveness of the pIL-10 carrier was interfered by pharmaceutical macrophage depletion using pre-injected clodronate liposome. When intravenously (IV) injected, our pIL-10 nanocarrier preferentially biodistributed in the injured muscle (owing to transiently decreased blood vessel integrity) and improved tissue repair. Unlike IL-10 protein (that was clinically abandoned), sustained IL-10 production by our nanocarrier dramatically reduced the degree of spleen enlargement and kidney inflammation, suggesting a favorable safety characteristic in vivo.