Adaptive Immune Response Signaling Is Suppressed in Ly6Chigh Monocyte but Upregulated in Monocyte Subsets of ApoE-/- Mice — Functional Implication in Atherosclerosis

RationaleInflammatory monocyte (MC) subset differentiation is a major feature in tissue inflammatory and atherosclerosis. The underlying molecular mechanism remains unclear.ObjectiveThis study aims to explore molecule targets and signaling which determinate immunological features in MC subsets.Methods and ResultsBlood Ly6Chigh and Ly6Clow MC subsets from control and ApoE-/- mice were isolated by flow cytometry sorting and subjected for bulk high-throughput RNA-sequencing. Intensive bioinformatic studies were performed by analyzing transcriptome through four pairs of comparisons: A) Ly6Chigh vs Ly6Clow in control mice; B) Ly6Chigh vs Ly6Clow in ApoE-/- mice; C) ApoE-/- Ly6Chigh vs control Ly6Chigh MC; D) ApoE-/- Ly6Clow vs control Ly6Clow MC. A total of 80 canonical pathways and 16 enriched pathways were recognized by top-down analysis using IPA and GSEA software, and further used for overlapping analysis. Immunological features and signaling were assessed on four selected functional groups, including MHCII, immune checkpoint, cytokine, and transcription factor (TF). Among the total 14578 significantly differentially expressed (SDE) genes identified though above four comparison, 1051 TF and 348 immunological genes were discovered. SDE immunological genes were matched with corresponding upstream SDE TF by IPA upstream analysis. Fourteen potential transcriptional axes were recognized to modulate immunological features in the Ly6C MC subset. Based on an intensive literature search, we found that the identified SDE immune checkpoint genes in Ly6Chigh MC are associated with pro-inflammatory/atherogenic balance function. Immune checkpoint genes GITR, CTLA4, and CD96 were upregulated in Ly6Clow MC from all mice and presented anti-inflammatory/atherogenic features. Six cytokine genes, including Ccl2, Tnfsf14, Il1rn, Cxcl10, Ccl9, and Cxcl2, were upregulated in Ly6Chigh MC from all mice and associated with pro-inflammatory/atherogenic feature. Cytokine receptor gene Il12rb2, Il1r1, Il27ra, Il5ra, Ngfr, Ccr7, and Cxcr5 were upregulated in Ly6Clow MC from all mice and presented anti-inflammatory/atherogenic features. MHCII genes (H2-Oa, H2-DMb2, H2-Ob, H2-Eb2, H2-Eb1, H2-Aa, and Cd74) were elevated in Ly6Clow MC from all mice. ApoE-/- augmented pro-atherogenic/inflammatory and antigen-presenting cells (APC) feature in both subsets due to elevated expression of cytokine genes (Cxcl11, Cntf, Il24, Xcl, Ccr5, Mpl, and Acvr2a) and MHCII gene (H2-Aa and H2-Ea-ps). Finally, we modeled immunological gene expression changes and functional implications in MC differentiation and adaptive immune response for MC subsets from control and ApoE-/- mice.ConclusionsLy6Chigh MC presented pro-inflammatory/atherogenic features and lower APC potential. Ly6Clow MC displayed anti-inflammatory/atherogenic features and higher APC potential. ApoE-/- confers upon both subsets with augmented pro-atherogenic/inflammatory function and APC potential.

Association of Pro-inflammatory Cytokine Gene Polymorphism with Meniere’s Disease in an Iranian Sample

Meniere’s disease (MD) is known as a rare chronic disorder of the inner ear with elevated serum levels of pro-inflammatory cytokines like tumor necrosis factor (TNF)-α, Interleukin (IL)-1, and IL-6. This study aims to evaluate genes polymorphism in some pro-inflammatory cytokines in a group of Iranian MD patients compared to the healthy controls. In this case-control study, 25 MD patients and 139 healthy controls were enrolled. DNA was extracted from blood samples, and single nucleotide polymorphisms were detected using polymerase chain reaction with sequence-specific primers assay. MD patients and controls were examined in terms of allele, genotype, and haplotype frequency of pro-inflammatory cytokine genes. Only the frequencies of alleles A/G at position -238 in the promoter of the TNF-α gene differed significantly between MD patients and healthy controls. G to A allele ratio was 23 and 3.6 in MD and controls, respectively. In individuals with MD, genotype GG was found to be significantly more prevalent at position -238 of the TNF-α gene promoter sequence. In addition, the heterozygote AG variant of -238 A/G TNF-α gene polymorphism was lower in MD patients than controls. Compared to the control group, the haplotype TNF- (-308, -238) AG was higher in MD patients, although not statistically significant. This is the first study that we know of that evaluates the frequencies of pro-inflammatory cytokine genes in an Iranian MD sample. This study shows the association between TNF-α and susceptibility to MD.

STW 5 Herbal Preparation Modulates Wnt3a and Claudin 1 Gene Expression in Zebrafish IBS-like Model

Aim: Irritable bowel syndrome (IBS) is a functional bowel disorder characterized by chronic abdominal pain and stool irregularities. STW 5 has proven clinical efficacy in functional gastrointestinal disorders, including IBS, targeting pathways that suppress inflammation and protect the mucosa. Wnt signaling is known to modulate NF-kβ-dependent inflammatory cytokine production. This sparked the idea of evaluating the impact of STW 5 on the expression of inflammatory-response and Wnt/β catenin-target genes in an IBS-like model. Main methods: We used zebrafish and dextran sodium sulfate (DSS) treatment to model IBS-like conditions in vivo and in vitro and examined the effects of subsequent STW 5 treatment on the intestines of DSS-treated fish and primary cultured intestinal and neuronal cells. Gross gut anatomy, histology, and the expression of Wnt-signaling and cytokine genes were analyzed in treated animals and/or cells, and in controls. Key findings: DSS treatment up-regulated the expression of interleukin-8, tumor necrosis factor-α, wnt3a, and claudin-1 in explanted zebrafish gut. Subsequent STW 5 treatment abolished both the macroscopic signs of gut inflammation, DSS-induced mucosecretory phenotype, and normalized the DSS-induced upregulated expression of il10 and Wnt signaling genes, such as wnt3a and cldn1 in explanted zebrafish gut. Under inflammatory conditions, STW 5 downregulated the expression of the pro-inflammatory cytokine genes il1β, il6, il8, and tnfα while it upregulated the expression of the anti-inflammatory genes il10 and wnt3a in enteric neuronal cells in vitro. Significance: Wnt signaling could be a novel target for the anti-inflammatory and intestinal permeability-restoring effects of STW 5, possibly explaining its clinical efficacy in IBS.

PLASMA Th1/Th2/Th17 CYTOKINE PROFILE AND CYTOKINE GENE POLYMORPHISMS (IL12B, IL13, IL31, IL33) IN ASTHMATIC CHILDREN: A MAGNETIC MULTIPLEX ASSAY

The study of the bronchial asthma pathogenesis is an urgent problem due to its high prevalence and often developing uncontrolled severe ashma, including in childhood. The first signs of asthma development tend to occur in childhood, which causes deterioration in the patient’s quality of life and early disability. Since BA is a genetically mediated process, the severity of the disease is assumed to depend on the presence of a specific allelic variant in the mediator (e.g. cytokines) genes involved in the BA pathogenesis. The aim of this study was to search for immunogenetic markers of severe asthma in Slavs children living in Krasnoyarsk city. The quantitative indicators of the Th1/Th2/Th17-cytokine profile in children with bronchial asthma (BA) with varying disease severity, depending on the polymorphism of cytokine genes, using the method of multiplex analysis (xMAP), were first determined. Changes in the cytokine background in BA patients fit into the concept that a percentage of neutrophilic endotype, which performs its functions through Th1 and Th17-lymphocytes in severe asthma, increases. In addition, the cytokine profile data depending on concomitant acute respiratory infections were obtained. There was an imbalance when analyzing the cytokine plasma level, with a tendency to maintain the protective functions of the immune system among patients in remission. Distribution of cytokine genes was obtained: allelic variants of IL12B rs321220*G, IL13 rs1800925*C, IL31 rs7977932*C and IL33 rs7044343*T are the most common in the population sampling from Krasnoyarsk. The probability of the genotype association of cytokine genes (IL12B, IL13, IL31, IL33) with the state of the immune system in bronchial asthma with varying disease severity in children was studied: a significant association of the TT genotype IL12B rs3212220 with a low concentration of IL-12B was presented. Our data obtained can be used along with the previously obtained immunogenetic markers of severe and uncontrolled asthma in children for patient-specific prognosis of the disease nature.

Inflammation modulates regeneration in the acute or chronically damaged zebrafish retina

Unlike mammals, zebrafish regenerate in response to retinal damage. Because microglia are activated by retinal damage, we investigated their role during regeneration following acute or chronic damage. At three weeks-post-fertilization (wpf), fish exhibiting NMDA-induced acute damage or cone photoreceptor-specific chronic degeneration, the gold rush (gosh) mutant, displayed reactive microglia and Müller glia proliferation. Retinas treated to inhibit the immune response lacked reactive microglia and possessed fewer PCNA-positive cells, while LPS treatment increased microglia and PCNA-labeled cells. NMDA-injured retinas upregulated the expression of il-1β and tnf-α pro-inflammatory cytokine genes, followed by increased expression of il-10 and arg1 anti-inflammatory/remodeling cytokine genes. An early and transiently TNF-α pro-inflammatory microglia population was identified in the NMDA-damaged retina. In contrast, gosh mutant retinas exhibited a mild increase of pro-inflammatory cytokine gene expression concurrently with a greater increased in anti-inflammatory/remodeling cytokine gene expression. Few TNF-α pro-inflammatory microglia were observed in the gosh retina. How inflammation regulates regeneration in zebrafish would provide important clues towards improving the therapeutic strategies for repairing injured mammalian tissues.