In vitro cytokine expression analysis by droplet microfluidics

Droplet microfluidics provides a miniaturized platform to conduct biological assays. We previously developed a droplet microfluidic chip assay for screening cancer cells against chemical drugs and chimeric antigen receptor T (CAR-T) cells, respectively. In this study, we investigated chip application on a cytokine expression assay using MCF7 breast cancer reporter cells engineered by fusing green fluorescent protein (GFP) to the C-terminus of endogenous interleukin-6 (IL6) gene. Combined tumor necrosis factor alpha (TNFalpha) treatment and serum-free medium starvation stimulated IL6-GFP expression and enhanced GFP fluorescence. Our data showed that on-chip assay recapitulates the cellular response in vitro, although absolute quantification of IL6 induction could not be accomplished. The demonstration of multi-timepoint IL6 expression analysis paves the way for our future study on tumor response to immune attack via cytokine signaling.

Resistance-exercise training attenuates LPS-induced astrocyte remodeling and neuroinflammatory cytokine expression in female Wistar rats

Neuroinflammation is an early detectable marker of mild cognitive impairment, the transition state between normal cognition and dementia. Resistance-exercise training can attenuate the cognitive decline observed in patients with mild cognitive impairment. However, the underlying mechanisms of resistance training effects are largely unknown. To further elucidate mechanisms of the known cognitive health benefits from resistance-exercise training, we tested if three weeks of resistance-exercise training could ameliorate lipopolysaccharide-induced neuroinflammation. Five-week-old female Wistar rats received intracerebroventricular injections of lipopolysaccharides to induce neuroinflammation and cognitive impairment. Rats then underwent three weeks of progressive ladder climbing to recapitulate resistance-exercise training in humans. Cognition was assessed towards the end of the training period by novelty object recognition testing. Neuroinflammation was measured one and 24-hours after the last resistance-exercise training workout. Resistance-exercise training ameliorated cognitive impairment, diminished lipopolysaccharide-induced neuroinflammatory cytokine expression, and attenuated astrocyte remodeling in the dentate gyrus 24-hours post exercise. Here, we provide evidence that the ladder-climbing model of resistance-exercise training in rats can improve cognition as early as three weeks. Additionally, these data support the hypothesis that resistance exercise can reduce lipopolysaccharide-induced neuroinflammation in the dentate gyrus.

The Novel Peptide AEDPPE Alleviates Trophoblast Cell Dysfunction Associated With Preeclampsia by Regulating the NF-κB Signaling Pathway

Background: Preeclampsia (PE) is a serious risk to the health of pregnant women and fetuses during pregnancy, and there is no effective treatment for this condition. Although many reports have confirmed the therapeutic effects of peptides in diseases, the role of peptides in PE remains poorly understood.Methods: A differentially expressed peptide in PE (AEDPPE) is derived from heat-shock protein beta-1 (HSPB1), amino acids 100 to 109 (DVNHFAPDEL), which we identified in a previous study. We synthesized AEDPPE and investigated its effect on HTR-8/SVneo cell function using a Cell Counting Kit-8, flow cytometric assay, and Transwell and wound-healing assays. Quantitative reverse transcription-PCR and ELISA were used to determine cytokine expression. Pull-down assay, mass spectrometry, Western blot analysis, and immunofluorescence were used to explore the potential targets and signaling pathways regulated by AEDPPE. Finally, we assessed the effect of AEDPPE in the lipopolysaccharide (LPS)-induced PE-like rat model.Results: AEDPPE significantly promoted the migration and invasion of HTR-8/SVneo cells, and it decreased the expression of interleukins 1 beta (IL-1β), interleukin 6 (IL-6), and interleukin 8 (IL-8). These functions performed by AEDPPE remained evident after injury to HTR-8/SVneo cells with tumor necrosis factor-alpha (TNF-α), and AEDPPE reversed the elevated sFlt-1/PlGF ratio induced by TNF-α. AEDPPE may exert these biological effects by binding to heat-shock protein 90β (HSP 90β) and, thus, affect the NF-κB signaling pathway. In an LPS-induced PE-like rat model, AEDPPE significantly improved PE symptoms and fetal rat outcomes.Conclusion: Our study showed that AEDPPE enhanced trophoblast migration and invasion and reduced inflammatory cytokine expression, and we hypothesized that these actions involved the NF-κB signaling pathway. The use of AEDPPE may thus develop into a novel modality in the treatment of PE.

Effects of post-weaning supplementation of immunomodulatory feed ingredient on circulating cytokines and microbial populations in programmed fed beef heifers

The objective was to determine the effects of an immunomodulatory feed ingredient following weaning on cytokine expression and fecal microbial populations of heifers. Commercial Angus heifers (n = 72) were weaned (227 ± 7 d of age), blocked by BW (n = 9 blocks) and randomly assigned to one of 2 pens per block. Pens within weight block (4 heifers/pen) were then randomly assigned to treatments. Heifers were fed twice daily from d 0-60 (to gain 0.75kg/day) and top-dressed with either 18g/heifer/d of the immunomodulatory feed ingredient (Celmanax; Arm and Hammer Animal Nutrition, Princeton, NJ, USA; CEL) or corn-germ meal (CON). Blood samples were collected on d 0, 15, 30, 45, 60 and fecal grab samples on d 0 of the feeding trial. After d 60, two heifers per pen (n=32) were randomly selected for a transportation challenge. Serum samples were collected at h 0, 4, 8, 12 and fecal grab samples at h -24, 0, 24 and 7d post-challenge. Blood samples were analyzed for interferonγ (IFNγ), interleukin-8 (IL-8), and haptoglobin (HP) using commercially available ELISA kits and qRT-PCR for genes of interest associated with cytokine expression. Fecal samples were enumerated for Clostridia and E. coli using selective media (≤ 5 isolates from each media/sample), tested to determine if they were C. perfringens or pathogenic E. coli, and then enriched for detection of Salmonella. Data was analyzed via ANOVA. During the feeding trial, HP was reduced (P = 0.018) in CEL compared to CON at d 15, 45, and 60, while IFNγ and IL-8 did not differ (P > 0.080) between treatments. All cytokines were decreased (P < 0.001) in CEL compared to CON during the challenge. During the feeding trial, HP mRNA was increased (P = 0.045) in CEL compared to CON at d 30 and 60. Similarly, IFNγ mRNA was increased (P = 0.040) in CEL compared to CON, however, other genes of interest did not differ (P > 0.172). Both C. perfringens and total E. coli counts were decreased (P = 0.036) in CEL compared to CON at 24h after the start of the transportation challenge. Clostridia and pathogenic E. coli counts did not differ (P = 0.941) between treatments. Total Clostridia and E. coli counts were increased (P < 0.014) 24h post-challenge. All microbial populations, except pathogenic E. coli, observed decreased (P ≤ 0.009) counts from 24h to 7d post-challenge. Overall, Celmanax supplementation decreased circulating cytokines, and altered microbial populations and gene expression, thus, may serve a role in preparing animals to better cope with immunological challenges.

Cytokine expression in the delayed-type hypersensitivity response of Peromyscus yucatanicus infected by Leishmania (Leishmania) mexicana that healed spontaneously / Expressão de citocinas na resposta de hipersensibilidade de tipo retardado de Peromyscus yucatanicus infectado por Leishmania (Leishmania) mexicana que curou espontaneamente

Peromyscus yucatanicus tem sido empregado como modelo para estudar a leishmaniose tegumentar causada por Leishmania (Leishmania) mexicana. No entanto, não há informações sobre a cura espontânea e a resposta imune associada nesses roedores. O objetivo deste trabalho foi analisar a expressão de citocinas na resposta de hipersensibilidade do tipo retardado de Peromyscus yucatanicus infectado por Leishmania (Leishmania) mexicana que cicatrizou espontaneamente. Peromyscus yucatanicus (n = 40) foram inoculados com 2.5 × 106 parasitas na cauda e a evolução foi registrada semanalmente até o aparecimento das lesões ativas (Grupo I: não cicatrizadas) e até a cicatrização espontânea (Grupo II: cicatrizadas). Um grupo controle foi injetado com meio RPMI-1640. A resposta de hipersensibilidade do tipo retardado (DTH) e as expressões de citocinas (IFN-γ, IL-10, TNF) foram determinadas. A cura espontânea foi observada em 65% (13/20) dos P. yucatanicus do Grupo II. O grupo curado desenvolveu uma forte reação DTH que foi significativamente maior do que o grupo controle. Às 24 h, o IFN-γ foi altamente expresso na reação DTH de ambos os camundongos não curados e curados. IL-10 foi maior em camundongos curados em comparação com o grupo controle, enquanto a expressão de TNF foi maior em camundongos não curados. Em 48 h, INF-γ foi altamente expresso em camundongos não curados. A cicatrização espontânea de lesões cutâneas em P. yucatanicus foi associada à expressão de citocinas imunorreguladoras (IL-10) e efetoras (IFN-γ) na resposta DTH.

Long noncoding RNA GSEC promotes neutrophil inflammatory activation by supporting PFKFB3-involved glycolytic metabolism in sepsis

Metabolic reprogramming is a hallmark of neutrophil activation in sepsis. LncRNAs play important roles in manipulating cell metabolism; however, their specific involvement in neutrophil activation in sepsis remains unclear. Here we found that 11 lncRNAs and 105 mRNAs were differentially expressed in three transcriptome datasets (GSE13904, GSE28750, and GSE64457) of gene expression in blood leukocytes and neutrophils of septic patients and healthy volunteers. After Gene Ontology biological process analysis and lncRNA–mRNA pathway network construction, we noticed that GSEC lncRNA and PFKFB3 were co-expressed and associated with enhanced glycolytic metabolism. Our clinical observations confirmed the expression patterns of GSEC lncRNA and PFKFB3 genes in neutrophils in septic patients. Performing in vitro experiments, we found that the expression of GSEC lncRNA and PFKFB3 was increased when neutrophils were treated with inflammatory stimuli. Knockdown and overexpression experiments showed that GSEC lncRNA was essential for mediating PFKFB3 mRNA expression and stability in neutrophil-like dHL-60 cells. In addition, we found that GSEC lncRNA-induced PFKFB3 expression was essential for mediating dHL-60 cell inflammatory cytokine expression. Performing mechanistic experiments, we found that glycolytic metabolism with PFKFB3 involvement supported inflammatory cytokine expression. In summary, our study uncovers a mechanism by which GSEC lncRNA promotes neutrophil inflammatory activation in sepsis by supporting glycolytic metabolism with PFKFB3.

Effects of Intermittent Hypoxia on Cytokine Expression Involved in Insulin Resistance

Sleep apnea syndrome (SAS) is a prevalent disorder characterized by recurrent apnea or hypoxia episodes leading to intermittent hypoxia (IH) and arousals during sleep. Currently, the relationship between SAS and metabolic diseases is being actively analyzed, and SAS is considered to be an independent risk factor for the development and progression of insulin resistance/type 2 diabetes (T2DM). Accumulating evidence suggests that the short cycles of decreased oxygen saturation and rapid reoxygenation, a typical feature of SAS, contribute to the development of glucose intolerance and insulin resistance. In addition to IH, several pathological conditions may also contribute to insulin resistance, including sympathetic nervous system hyperactivity, oxidative stress, vascular endothelial dysfunction, and the activation of inflammatory cytokines. However, the detailed mechanism by which IH induces insulin resistance in SAS patients has not been fully revealed. We have previously reported that IH stress may exacerbate insulin resistance/T2DM, especially in hepatocytes, adipocytes, and skeletal muscle cells, by causing abnormal cytokine expression/secretion from each cell. Adipose tissues, skeletal muscle, and the liver are the main endocrine organs producing hepatokines, adipokines, and myokines, respectively. In this review, we focus on the effect of IH on hepatokine, adipokine, and myokine expression.