Substance P promotes epidural fibrosis via induction of type 2 macrophages.

In response to spinal surgery, neurons secrete a large amount of substance P into the epidural area. Substance P is involved in macrophage differentiation and fibrotic disease. However, the specific roles and mechanisms of substance P in epidural fibrosis remain unclear. In this study, we established a mouse model of L1-L3 laminectomy and found that dorsal root ganglion neurons and the macrophages infiltrating into the wound area released sphingolipids. In vitro experiments revealed that type 1 macrophages secreted substance P, which promoted differentiation of type 1 macrophages towards a type 2 phenotype. High-throughput mRNA-seq analysis revealed that the sphingolipid metabolic pathway may be involved in the regulation of type 2 macrophages by substance P. Specifically, sphingomyelin synthase 2, a component of the sphingolipid metabolic pathway, promoted M2 differentiation in substance P-treated macrophages, while treating the macrophages with LY93, a sphingomyelin synthase 2 inhibitor, suppressed M2 differentiation. In addition, substance P promoted the formation of neutrophil extracellular traps, which further boosted M2 differentiation. Blocking substance P with the neurokinin receptor 1 inhibitor RP67580 decreased the number of M2 macrophages in the wound area after spinal surgery and alleviated epidural fibrosis, as evidenced by decreased fibronectin, α-smooth muscle actin, and collagen I in the scar tissue. These results demonstrated that substance P promotes M2 macrophage differentiation in epidural fibrosis via sphingomyelin synthase 2 and neutrophil extracellular traps. These findings provide a novel strategy for the treatment of epidural fibrosis.

Coexpression of Helios in Foxp3+ Regulatory T Cells and Its Role in Human Disease.

Regulatory T cells (Tregs) expressing the Foxp3 transcription factor are indispensable for the maintenance of immune system homeostasis. Tregs may lose Foxp3 expression or be reprogrammed into cells that produce proinflammatory cytokines, for example, Th1-like Tregs, Th2-like Tregs, Th17-like Tregs, and Tfh-like Tregs. Accordingly, selective therapeutic molecules that manipulate Treg lineage stability and/or functional activity might have the potential to improve aberrant immune responses in human disorders. In particular, the transcription factor Helios has emerged as an important marker and modulator of Tregs. Therefore, the current review focuses on recent findings on the expression, function, and mechanisms of Helios, as well as the patterns of Foxp3+ Tregs coexpressing Helios in various human disorders, in order to explore the potential of Helios for the improvement of many immune-related diseases. The studies were selected from PubMed using the library of the Nanjing Medical University in this review. The findings of the included studies indicate that Helios expression stabilizes the phenotype and function of Foxp3+ Tregs in certain inflammatory environments. Further, Tregs coexpressing Helios and Foxp3 were identified as a specific phenotype of stronger suppressor immune cells in both humans and animal models. Importantly, there is ample evidence that Helios-expressing Foxp3+ Tregs are relevant to various human disorders, including connective tissue diseases, infectious diseases, solid organ transplantation-related immunity, and cancer. Thus, Helios+Foxp3+CD4+ Tregs could be a valuable target in human diseases, and their potential should be explored further in the clinical setting.

Downregulation of miR-4772-3p promotes enhanced regulatory T cell capacity in malignant pleural effusion by elevating Helios levels.

BACKGROUND: Malignant pleural effusion (MPE) is a complicated condition of patients with advanced tumors. Further dissecting the microenvironment of infiltrated immune cells and malignant cells are warranted to understand the immune-evasion mechanisms of tumor development and progression. METHODS: The possible involvement of microRNAs (miRNAs) in malignant pleural fluid was investigated using small RNA sequencing. Regulatory T cell (Treg) markers (CD4, CD25, forkhead box P3), and Helios (also known as IKAROS Family Zinc Finger 2 [IKZF2]) were detected using flow cytometry. The expression levels of IKZF2 and miR-4772-3p were measured using quantitative real-time reverse transcription polymerase chain reaction. The interaction between miR-4772-3p and Helios was determined using dual-luciferase reporter assays. The effects of miR-4772-3p on Helios expression were evaluated using an in vitro system. Correlation assays between miR-4772-3p and functional molecules of Tregs were performed. RESULTS: Compared with non-malignant controls, patients with non-small cell lung cancer had an increased Tregs frequency with Helios expression in the MPE and peripheral blood mononuclear cells. The verified downregulation of miR-4772-3p was inversely related to the Helios Tregs frequency and Helios expression in the MPE. Overexpression of miR-4772-3p could inhibit Helios expression in in vitro experiments. However, ectopic expression of Helios in induced Tregs reversed the effects induced by miR-4772-3p overexpression. Additionally, miR-4772-3p could regulate Helios expression by directly targeting IKZF2 mRNA. CONCLUSION: Downregulation of miR-4772-3p, by targeting Helios, contributes to enhanced Tregs activities in the MPE microenvironment.

[Screening and identification of differential expression genes related to stress fracture by cDNA microarray assay].

OBJECTIVE: To evaluate the differentially expressed genes between the Stress fracture (SF) cases and controls. METHODS: Total RNA was extracted and purified from peripheral blood sample of 3 SF cases and 3 controls who conducted a 1:1 matched case-control study, then used for Human Genome Array analysis. The hybridization data were analyzed using SAM software. Parts of these genes were analyzed and identified by real-time PCR. RESULTS: Upregulated and downregulated genes were 22 and 1, respectively. Thus the highest ratio and most significant cytokine was tumor necrosis factor receptor superfamily, member 10c (TNFRSF10C). The result of real-time PCR shows that TNFRSF10C was over-expressed in 3 cases and low-expressed in 1 case. CONCLUSION: Obvious difference exists in gene expression between SF cases and controls, showing there may be a lot of genes involving in the occurrence and development of SF. Meanwhile, the identification of the specific genes is helpful for biomechanics study, early diagnosis and screening of SF.

Targeted NF-kappaB inhibition of asthmatic serum-mediated human monocyte-derived dendritic cell differentiation in a transendothelial trafficking model.

Transendothelial trafficking model mimics in vivo differentiation of monocytes into dendritic cells (DC). The serum from patients with systemic lupus erythematosus promotes the differentiation of monocytes into mature DC. We have shown that selective inhibition of NF-kappaB by adenoviral gene transfer of a novel mutated IkappaBalpha (AdIkappaBalphaM) in DC contributes to T cell tolerance. Here we demonstrated for the first time that asthmatic serum facilitated human monocyte-derived DC (MDDC) maturation associated with increased NF-kappaB activation in this model. Furthermore, selective blockade of NF-kappaB by AdIkappaBalphaM in MDDC led to increased apoptosis, and decreased levels of CD80, CD83, CD86, and IL-12 p70 but not IL-10 in asthmatic serum-stimulated MDDC, accompanied by reduced proliferation of T cells. These results suggest that AdIkappaBalphaM-transferred MDDC are at a more immature stage which is beneficial to augment the immune tolerance in asthma.

Effects of adenoviral gene transfer of mutated IkappaBalpha, a novel inhibitor of NF-kappaB, on human monocyte-derived dendritic cells.

AIM: To investigate the effects of adenoviral gene transfer of IkappaBalpha mutant (IkappaBalphaM), a novel inhibitor of nuclear factor kappaB (NF-kappaB), on apoptosis, phenotype and function of human monocyte-derived dendritic cells (DC). METHODS: Monocytes, cocultured with granulocyte/macrophage colony-stimulating factor (GM-CSF; 900 ng/mL) and interleukin (IL)-4 (300 ng/mL) for 5 d, followed by stimulation with lipopolysaccharide (LPS; 100 ng/mL) for 2 d differentiated into mature DC. Monocytes were either left untransfected or were transfected with AdIkappaBalphaM or AdLacZ. The transcription and expression of the IkappaBalphaM gene, and the inhibitory effect of IkappaBalphaM on tumor necrosis factor (TNF)-alpha-induced NF-kappaB activation in mature DC were detected by polymerase chain reaction (PCR), reverse transcription-polymerase chain reaction (RT-PCR), Western blot analysis, and electrophoretic mobility shift assays, respectively. The phenotype, apoptosis, IL-12 secretion level of DC, and ability to stimulate the proliferation of T cells were determined by flow cytometry, enzyme-linked immunosorbent assay and mixed leukocyte reaction. RESULTS: PCR and RT-PCR were used to detect a unique 801 bp band in AdIkappaBalphaM-transfected mature DC, and also a dose- and time-dependent expression of the IkappaBalphaM gene, which peaked at a multiplicity of infection of 100 pfu/cell and at 48 h. Furthermore, AdIkappaBalphaM significantly suppressed the TNF-alpha-induced NF-kappaB activation, augmented apoptosis, downregulated CD80, CD83, and CD86 surface molecules, IL-12 secretion levels and the ability to stimulate the proliferation of T cells in mature DC. CONCLUSION: AdIkappaBalphaM effectively transfected and potently inhibited NF-kappaB activation in monocyte-derived mature DC. Overexpression of the IkappaBalphaM gene in mature DC may contribute to T-cell immunosuppression through induction of DC apoptosis and downregulation of B7 molecules, providing a potential strategy for future DC-based immunotherapy of asthma.