Integrated analysis of single-cell sequencing and machine learning identifies a signature based on monocyte/macrophage hub genes to analyze the intracranial aneurysm associated immune microenvironment.

Monocytes are pivotal immune cells in eliciting specific immune responses and can exert a significant impact on the progression, prognosis, and immunotherapy of intracranial aneurysms (IAs). The objective of this study was to identify monocyte/macrophage (Mo/MΦ)-associated gene signatures to elucidate their correlation with the pathogenesis and immune microenvironment of IAs, thereby offering potential avenues for targeted therapy against IAs. Single-cell RNA-sequencing (scRNA-seq) data of IAs were acquired from the Gene Expression Synthesis (GEO) database. The significant infiltration of monocyte subsets in the parietal tissue of IAs was identified using single-cell RNA sequencing and high-dimensional weighted gene co-expression network analysis (hdWGCNA). The integration of six machine learning algorithms identified four crucial genes linked to these Mo/MΦ. Subsequently, we developed a multilayer perceptron (MLP) neural model for the diagnosis of IAs (independent external test AUC=1.0, sensitivity =100%, specificity =100%). Furthermore, we employed the CIBERSORT method and MCP counter to establish the correlation between monocyte characteristics and immune cell infiltration as well as patient heterogeneity. Our findings offer valuable insights into the molecular characterization of monocyte infiltration in IAs, which plays a pivotal role in shaping the immune microenvironment of IAs. Recognizing this characterization is crucial for comprehending the limitations associated with targeted therapies for IAs. Ultimately, the results were verified by real-time fluorescence quantitative PCR and Immunohistochemistry.

Exploring the causal role of immune cells in cerebral aneurysm through single-cell transcriptomics and Mendelian randomization analysis.

Cerebral aneurysm (CA) represents a significant clinical challenge, characterized by pathological dilation of cerebral arteries. Recent evidence underscores the crucial involvement of immune cells in CA pathogenesis. This study aims to explore the complex interplay between immune cells and CA formation. We analyzed single-cell RNA sequencing data from the GSE193533 dataset, focusing on unruptured CA and their controls. Comprehensive cell-type identification and pseudo-time trajectory analyses were conducted to delineate the dynamic shifts in immune cell populations. Additionally, a two-sample Mendelian randomization (MR) approach was employed to investigate the causal influence of various immunophenotypes on CA susceptibility and the reciprocal effect of CA formation on immune phenotypes. Single-cell transcriptomic analysis revealed a progressive loss of vascular smooth muscle cells (VSMCs) and an increase in monocytes/macrophages (Mo/MΦ) and other immune cells, signifying a shift from a structural to an inflammatory milieu in CA evolution. MR analysis identified some vital immunophenotypes, such as CD64 on CD14+ CD16+ monocytes (OR: 1.236, 95% CI: 1.064-1.435, P = 0.006), as potential risk factors for CA development, while others, like CD28- CD8br %CD8br (OR: 0.883, 95% CI: 0.789-0.988, P = 0.030), appeared protective. Reverse MR analysis demonstrated that CA formation could modulate specific immunophenotypic expressions, highlighting a complex bidirectional interaction between CA pathology and immune response. This study underscores the pivotal role of immune cells in this process through the integration of single-cell transcriptomics with MR analysis, offering a comprehensive perspective on CA pathogenesis, and potentially guiding future therapeutic strategies targeting specific immune pathways.

Effects of regulatory B cells on intracranial aneurysms by mediating IL-1ß/IL-1R pathways.

The purpose of this study was to explore the effects of regulatory B-cells (Breg) on intracranial aneurysms by mediating IL-1ß/IL-1R pathways.  The study involved 60 patients undergoing angiography in a hospital from January to June 2022, divided into two groups: 30 with intracranial aneurysms (observation group) and 30 without (control group). Researchers extracted peripheral blood mononuclear cells (PBMC) to analyze the proportion of CD19+CD24hiCD38hiB cells using flow cytometry. These cells, along with T-cells and regulatory T-cells (Treg), were isolated through magnetic bead cell sorting. Following co-culture, the proliferation of T-cells and their related secretory factors were assessed. Additionally, Breg cells, treated with an IL-1R receptor blocker or IL-1R expression adenovirus, were studied to evaluate the levels of IL-10 and TGF-ß. In the study, the observation group showed lower levels of CD19+CD24hiCD38hiB cells, IL-10, and TGF-ß in PBMC than the control group (P<0.05). T-cell proportions were similar in both groups pre and post co-culture (P>0.05). Post co-culture, IFN-γ decreased while IL-4 increased in both groups. The observation group had higher IFN-γ and lower IL-4 than the control group (P<0.05). TNF-α in CD8+T cells, and granzyme B and perforin mRNA levels decreased post co-culture but were higher in the observation group (P<0.05). IL-10 and TGF-ß in Treg cells increased in both groups post co-culture but were lower in the observation group (P<0.05). The observation group also had fewer CD19+IL-1R+IL-10+B cells (P<0.05). After IL-1R blocker addition, IL-10 and TGF-ß in the supernatant decreased in the observation group (P<0.05). Following transfection, IL-1 and TGF-ß levels increased compared to the blank group (P<0.05). The function of peripheral blood CD19+CD24hiCD38hiB cells is impaired in patients with intracranial aneurysms, which may be related to IL-1ß/IL-1R pathways disorder.

Understanding the genetics of intracranial aneurysms: A primer.

The genetics of intracranial aneurysms is complex. Much work has been done looking at the extracellular matrix surrounding cerebral vasculature as well as the role of matrix metalloproteinases. This comprehensive review summarizes what is known to date about the important genetic components that predispose to aneurysm formation and critically discusses the published findings. We discuss promising pre-clinical models of aneurysm formation and subarachnoid hemorrhage, and highlight avenues for future discovery, while considering limitations in the research to date. This review will further serve as a comprehensive reference guide to understand the genetic underpinnings for aneurysm pathophysiology and act as a primer for further investigation.

Identification and Validation of TREM2 in Intracranial Aneurysms.

Background: Intracranial aneurysm (IA) is a cerebrovascular disease that seriously endangers human heath and life. However, the pathogenesis of IA has not been clarified. Objective: In this study, we explored the role of the triggering receptor expressed on myeloid cells-2 (TREM2) gene to explore a novel mechanism underlying IA. Methods: First, we verified the role of the candidate gene, TREM2 in a modified mouse model of IA. Second, we verified elevated expression of TREM2 using the Gene Expression Omnibus (GEO) database (GSE54083 and GSE75436) and developed protein interaction (PPI) network analysis using the top one hundred DEGs from GSE75436 dataset. Finally, we predicted a likely mechanism by which TREM2 is involved in the pathology of IA using single-gene Gene Set Enrichment Analysis (GSEA). Results: The expression of TREM2 and inflammatory factors was significantly increased in the modified mouse IA model, and showed a positive correlation. Elevated expression of TREM2 was also found in IA patients tissues from the GSE54083 and GSE75436 data sets. PPI network analyses suggested that the DEGs were involved in a variety of inflammatory processes. The GSEA results suggest that TREM2 may participate in IA progression by regulating macrophage function. Conclusion: TREM2 is highly expressed in both human and mouse IA tissues, and may participate in IA progression by regulating macrophage function and inflammatory factor expression. The molecular mechanism of TREM2 involvement in the IA process can be further studied using our modified mouse IA model.

Construction and Validation of a New Model for the Prediction of Rupture in Patients with Intracranial Aneurysms.

BACKGROUND: Despite progress in the detection of biological molecules that contribute to intracranial aneurysm (IA) development, many pathophysiological mechanisms remain unclear, particularly with regard to predicting IA rupture. In this study, we aimed to identify hub genes and construct a new model to predict IA rupture. METHODS: Four datasets (62 ruptured IAs, 16 unruptured IAs, and 31 normal controls) were downloaded from the Gene Expression Omnibus. Differentially expressed genes (DEGs) were identified between the IAs and normal controls. All overlapping genes were analyzed using weighted gene co-expression network analysis. Functional enrichment analyses were performed using key modules. We then intersected the key module genes with DEGs. Protein-protein interaction networks were assessed to identify key hub genes. Least absolute shrinkage and selection operator logistic regression analysis was performed to construct a prediction model. A receiver operating characteristic curve was constructed to evaluate the reliability of the scoring system. RESULTS: After intersection and normalization, 433 DEGs were identified and 15,388 genes were selected for weighted gene co-expression network analysis. The black module with 1145 genes exhibited the highest correlation with IA rupture. Many potential mechanisms are involved, such as the inflammatory response, innate immune response, extracellular exosome, and extracellular space. Thirty hub genes were selected from the protein-protein interaction, and 4 independent risk genes, TNFAIP6, NCF2, OSM, and IRAK3, were identified in the least absolute shrinkage and selection operator logistic regression model. CONCLUSIONS: Our prediction model not only serves as a useful tool for assessing the risk of IA rupture, but the key genes identified herein could also serve as biomarkers and therapeutic targets.

Mast Cell Promotes the Development of Intracranial Aneurysm Rupture.

BACKGROUND AND PURPOSE: Inflammation has emerged as a key component of the pathophysiology of intracranial aneurysms. Mast cells have been detected in human intracranial aneurysm tissues, and their presence was associated with intramural microhemorrhage and wall degeneration. We hypothesized that mast cells play a critical role in the development of aneurysmal rupture, and that mast cells can be used as a therapeutic target for the prevention of aneurysm rupture. METHODS: Intracranial aneurysms were induced in adult mice using a combination of induced systemic hypertension and a single injection of elastase into the cerebrospinal fluid. Aneurysm formation and rupture were assessed over 3 weeks. Roles of mast cells were assessed using a mast cell stabilizer (cromolyn), a mast cell activator (C48/80), and mice that are genetically lacking mature mast cells (KitW-sh/W-sh mice). RESULTS: Pharmacological stabilization of mast cells with cromolyn markedly decreased the rupture rate of aneurysms (80% versus 19%, n=10 versus n =16) without affecting the aneurysm formation. The activation of mast cells with C48/80 significantly increased the rupture rate of aneurysms (25% versus 100%, n=4 versus n=5) without affecting the overall rate of aneurysm formation. Furthermore, the genetic deficiency of mast cells significantly prevented aneurysm rupture (80% versus 25%, n=10 versus n=8, wild-type versus KitW-sh/W-sh mice). CONCLUSIONS: These results suggest that mast cells play a key role in promoting aneurysm rupture but not formation. Stabilizers of mast cells may have a potential therapeutic value in preventing intracranial aneurysm rupture in patients.

Clinical Relevance of Changes in Peripheral Blood Cells After Intracranial Aneurysm Rupture.

BACKGROUND: The rupture of an intracranial aneurysm (IA) causes a systemic response that involves an immune/inflammatory reaction. We sought to characterize the systemic response to IA rupture. METHODS: We included 19 patients in the acute phase of IA rupture and 20 control subjects. Flow cytometry was used to analyze alterations in the level of mononuclear leukocytes. Cell-related parameters, including the neutrophil-to-lymphocyte ratio (NL-R), lymphocyte-to-monocyte ratio (LM-R), platelet-to-lymphocyte ratio (PL-R), and systemic immune-inflammation index (SII), were calculated, and the relationship between the analyzed hematological parameters and clinical status was investigated. RESULTS: Patients with ruptured IAs presented with significantly higher white blood cells (WBC) and neutrophil counts but lower lymphocyte counts than control subjects. NL-R and SII values were higher and the LM-R was lower in the acute phase after IA rupture. Analyzing the severity of clinical status and the outcome of patients with subarachnoid hemorrhage, we found that patients with poor clinical status, as measured by the Glasgow Coma Scale (GCS) and the Hunt and Hess scale, had significantly lower lymphocyte counts and higher NL-R, PL-R and SII values than those with good clinical status. Additionally, patients with lower GCS scores presented a lower proportion of CD3+CD4-CD8- cells. Worse outcomes assessed at discharge were associated with lower lymphocyte counts but higher PL-R values. CONCLUSIONS: The current study pointed to the significance of systemic immune and inflammatory responses after IA rupture and the potential clinical utility of hematological parameters, which can be easily calculated. In particular, the role of DN T cells and the significance of the SII as a marker related to clinical status should be further investigated.

Immune Characterization in Aneurysmal Subarachnoid Hemorrhage Reveals Distinct Monocytic Activation and Chemokine Patterns.

The pathophysiology of delayed cerebral ischemia (DCI) after aneurysmal subarachnoid hemorrhage (aSAH) is incompletely understood. Intrathecal activation of inflammatory immune cells is suspected to play a major role for the induction of DCI. The aim of this study is to identify immune cell subsets and mediators involved in the pathogenesis of DCI. We prospectively collected blood and CSF from 25 patients with aSAH at early and late time points. We performed multicolor flow cytometry of peripheral blood and CSF, analyzing immune cell activation and pro-inflammatory cyto- and chemokines. In addition to the primary immune analysis, we retrospectively analyzed immune cell dynamics in the CSF of all our SAH patients. Our results show an increased monocyte infiltration secondary to aneurysm rupture in patients with DCI. Infiltrating monocytes are defined by a non-classical (CD14dim CD16+) phenotype at early stages. The infiltration is most likely triggered by the intrathecal immune activation. Here, high levels of pro-inflammatory chemokines, such as CXCL1, CXCL9, CXCL10, and CXCL11, are detected. The intrathecal cellular activation profile of monocytes was defined by upregulation of CD163 and CD86 on monocytes and a presumable later differentiation into antigen-presenting plasmacytoid dendritic cells (pDCs) and hemosiderophages. Peripheral immune activation was reflected by CD69 upregulation on T cells. Analysis of DCI prevalence, Hunt and Hess grade, and clinical outcome correlated with the degree of immune activation. We demonstrate that monocytes and T cells are activated intrathecally after aSAH and mediate a local inflammatory response which is presumably driven by chemokines. Our data shows that the distinct pattern of immune activation correlates with the prevalence of DCI, indicating a pathophysiological connection to the incidence of vasospasm.

Characteristics of circulating monocytes at baseline and after activation in patients with intracranial aneurysm.

Intracranial aneurysm (IA) is a bulging of blood vessels around the brain that is often asymptomatic but may cause severe complications and death if ruptured. Macrophage-mediated immune responses can contribute to the development of IA. During homeostasis and inflammation, circulating monocytes can infiltrate the vasculature, where they develop into macrophages, and modulate immune responses. Based on the expression of CD14 and CD16, total circulating monocytes can be distinguished into three main subsets, including the CD14+CD16- classical monocytes, the CD14+CD16+ intermediate monocytes, and the CD14loCD16++ non-classical monocytes. In this study, we found that frequencies of CD14+CD16- classical monocytes were significantly lower in IA patients than in healthy controls, while the frequencies of CD14+CD16+ intermediate monocytes and CD14loCD16++ non-classical monocytes were significantly higher in IA patients than in healthy controls. The frequencies of CD14+CD16+ intermediate monocytes were further elevated in IA-ruptured patients compared to those in IA-unruptured patients. Compared to classical monocytes, intermediate monocytes and non-classical monocytes presented higher TNF-α and IL-1ß expression. When cocultured with autologous naive CD4 T cells, intermediate and non-classical monocytes preferentially promoted the expression of TBX21 and RORC over the expression of FOXP3 in CD4 T cells. Inhibition of TNF-α and IL-1ß slightly reduced TBX21 expression and markedly reduced RORC expression, and at the same time significantly increased FOXP3 expression in CD4 T cells. Overall, this study demonstrated that the monocytes were dysregulated in IA patients in a manner that favored the development of proinflammatory responses.

Tumor-associated macrophage-derived exosomal microRNA-155-5p stimulates intracranial aneurysm formation and macrophage infiltration.

Tumor-associated macrophages (TAMs) play a regulatory role in inflammation and cancer. Exosomes derived from macrophages carrying microRNAs (miRNAs or miRs) are of great value for cancer therapy. Gremlin 1 (GREM1), a member of the antagonists of secreted bone morphogenetic protein, has been implicated in the pathophysiology of multiple diseases or cancers. Based on the predictions of miRNA-mRNA interaction, GREM1 was found to be a target gene of miR-155-5p. Here, the present study aims to explore the role of TAM-derived exosomal miR-155-5p by regulating GREM1 in intracranial aneurysm (IA). The collected results showed that GREM1 was down-regulated in IA, while miR-155-5p was up-regulated in TAM-derived exosomes. Smooth muscle cells (SMCs) were co-cultured with TAMs or exposed to exosomes derived from TAMs transfected with either miR-155-5p mimic or miR-155-5p inhibitor for exploring their roles in proliferation and migration of SMCs in vitro. Accordingly, in vitro experiments showed that TAM-derived exosomal miR-155-5p could promote proliferation and migration of SMCs by targeting GREM1. The effects of TAM-derived exosomal miR-155-5p on IA formation and TAM activation and infiltration by regulation of GREM1 in vivo were measured in IA rats injected with exosomes or those from TAMs transfected with miR-155-5p inhibitor. In vivo experimental results consistently confirmed that TAM-derived exosomes carrying miR-155-5p promoted IA formation and TAM activation and infiltration. In conclusion, TAM-derived exosomal miR-155-5p promotes IA formation via GREM1, which points to miR-155-5p as a possible therapeutic target for IA.

ß-Sitosterol Attenuates the Intracranial Aneurysm Growth by Suppressing TNF-α-Mediated Mechanism.

BACKGROUND: Onset of inflammation associated with increased extracellular matrix degradation of vascular walls in the neuronal area is the pathophysiology of cerebral aneurysms. It has been documented well that ß-sitosterol has protective effects on various brain-related diseases independent of their lipid-lowering effects; the current work was framed to examine the effect of ß-sitosterol on CA progression. MATERIALS AND METHODS: To study whether ß-sitosterol has a suppressive effect on the growth of CA, ß-sitosterol administration started prior to aneurysm induction. CA was induced in Wistar male rats with or without oral administration of ß-sitosterol. The expression of chemokines and inflammatory cytokines, tumor necrosis factor-α (TNF-α), interleukin (IL)-8, IL-1ß, IL-17, IL-6, matrix metalloproteinases (MMP)-2 and -9, was elucidated by ELISA and RT-PCR. RESULTS: Rats treated with ß-sitosterol exhibited a significant reduction in aneurysmal size compared with control rats. In addition, ß-sitosterol administration reduced the expression of chemokines and inflammatory cytokines, while gelatin zymography data revealed declined activity of MMP-2 and -9 in aneurismal walls. Furthermore, the levels of cytokines were significantly reduced in ß-sitosterol-administered rats compared to CA rats. CONCLUSIONS: Treatment with ß-sitosterol suppresses the development of CA by inhibiting inflammatory reactions including TNF-α and thus ß-sitosterol can be a suggestive candidate for the prevention of CA treatment and progression.

Tanshinone IIA attenuates cerebral aneurysm formation by inhibiting the NF­κB­mediated inflammatory response.

The inflammatory response plays a vital role in cerebral aneurysm (CA) formation and progression. Tanshinone  IIA (Tan IIA) is one of the major active components of Chinese medicine Danshen (Salvia miltiorrhiza Bunge) and is widely used for the treatment of cardiovascular diseases, due to its anti­inflammatory effects. The aim of the present study was to investigate whether Tan IIA can attenuate CA formation in rat models, and determine its underlying mechanisms. CAs were induced in rats surgically and through high­salt diet treatments. The Tan IIA­treated group displayed relatively mild symptoms, as compared with the control group. Tan IIA treatment reduced macrophage infiltration and nuclear factor (NF)­κB activation in aneurysmal walls. Next, lipopolysaccharide (LPS)­stimulated RAW 264.7 murine macrophage cells were used to examine the anti­inflammatory effects of Tan IIA on macrophages. It was found that Tan IIA reversed LPS­induced differentiation of RAW 264.7 cells and suppressed NF­κB pathway activation. In conclusion, these findings demonstrated that Tan IIA can suppress CA formation by inhibiting inflammatory responses in macrophages.

Histological analysis of infiltrating macrophages in the cerebral aneurysm walls.

A series of recent evidences suggested activated macrophages have broadly two distinct forms that possess opposite functions for the process of inflammation: classically activated macrophages (M1/kill macrophages) and alternatively activated macrophages (M2/repair macrophages) according to their functions and expression markers. To elucidate what roles those two phenotypes of macrophages play in the evolution of cerebral aneurysm, the presence of macrophages inside the aneurysm walls was assessed with an immunohistochemical approach. The portions of the aneurysm domes deflated after neck clipping were utilized for the further histological examinations, including immunostainings with five antibodies to identify macrophage subpopulations. In this study, contrary to the previous reports, the following various ratios of subtypes were observed in the aneurysm walls: M1 > M2 (2 cases), M1 < M2 (2 cases), M1 = M2 (3 cases). While M1-like macrophages have been typically regarded as a main driver of the degenerating process, these surprisingly richer presences of M2-like macrophages in the aneurysm walls suggests that an unrecognized biological process might be in play in aneurysm development.

Current Understanding of the Molecular Mechanism between Hemodynamic- Induced Intracranial Aneurysm and Inflammation.

Intracranial aneurysms (IA) are a huge threat to human health, with a global incidence rate of 0.65-8.4%. Although the microsurgical and interventional techniques have made profound progression in treating IA, the relatively high rate of complications and recurrence are still not satisfactory. Thus, there is a need to elucidate its molecular mechanism. Numerous studies have identified the close relationship between hemodynamic-induced inflammation and development of IA. Indeed, the dysfunction of endothelial cells, smooth muscle cells, macrophages and lymphocytes, as well as their secreted cytokines, collectively contribute to the formation, growth and rupture of IA. Furthermore, the immune system has also been identified to participate in the development of IA. This review will explore the mechanisms of various inflammatory cells and significant cytokines, providing a new perspective in the clinical treatment of IA.

Regulatory T cells demonstrate significantly increased functions following stimulation with IL-2 in a Tim-3-dependent manner in intracranial aneurysms.

The formation of intracranial aneurysm (IA) is associated with the destruction of various cellular and structural components, which induces pathogenic inflammatory responses that further propagate tissue damage. The regulatory immune system can suppress exacerbated inflammation and offer tissue protection; however, previous studies by others and us have demonstrated that the regulatory T (Treg) cells were functionally impaired in IA patients. Hence, strategies that can improve Treg function in IA patients should be investigated. Based on our previous finding that IL-2 strongly elevated the expression of the checkpoint molecule Tim-3 in Treg cells, we examined the effect of IL-2 in the function of Treg cells from IA patients. External IL-2 significantly improved the proliferation of Treg cells, increased the expression of CTLA-4 and LAG-3, and enhanced Treg-mediated suppression of conventional T cell (Tconv) proliferation. Importantly, compared to the Tim-3- Treg cells, the Tim-3+ Treg cells presented comparable proliferation capacity, but significantly greater expressions of CTLA-4 and LAG-3 and significantly higher capacity to suppress Tconv proliferation. In addition, blocking Tim-3 abrogated IL-2-mediated enhancement of Tim-3+ Treg cells. We then investigated the IL-2 level in IA patients, and found that although IA patients and healthy controls presented similar serum IL-2 concentration, the concentrations of IL-1ß and TNF-α were significantly higher in IA patients than in healthy controls, signaling a relative reduction in IL-2 abundance. Together, we found that IL-2 could significantly enhance the function of Treg cells from IA patients in a Tim-3-dependent manner.

Patients with intracranial aneurysms presented defects in regulatory T cells, which were associated with impairment in Tim-3 upregulation.

Pathogenic inflammation contributes to aneurysm formation by mediating the destruction of the endothelium and the extracellular matrix and promoting pathogenic proliferation of smooth muscle cells. In mouse models, tolerance-inducing T regulatory (Treg) cells could significantly reduce the incidence and severity of aneurysms. Hence, it should be investigated why in human intracranial aneurysm (IA) patients, Treg cells failed to provide protection against aneurysm formation. In this study, the frequency and function of Treg cells in IA patients were examined. The frequency of Foxp3+ Treg cells was significantly lower in IA patients than in healthy controls. This downregulation was only specific to the Treg subset of CD4+ T cells, as the frequency of total CD4+ T cell was increased in IA patients. Subsequently, we found that the expressions of Treg-associated molecules, including Foxp3, CTLA-4, TGF-ß, and IL-10, were significantly lower in Foxp3+ Treg cells from IA patients than in Foxp3+ Treg cells from healthy controls. In both healthy controls and IA patients, Foxp3+ Treg cells were distinguished into a more potent Tim-3+ subset and a less potent Tim-3- subset. The Tim-3+ subset of Foxp3+ Treg cells was significantly reduced in IA patients. Signaling via IL-2, IL-7, IL-15 and IL-21 was shown to promote Tim-3 upregulation in CD4+ and CD8+ T cells. Interestingly, we found that Tim-3 could be upregulated in Treg cells via the same mechanism, but compared to the Treg cells from healthy controls, the Treg cells from IA patients presented defects in Tim-3 upregulation upon cytokine stimulation. Together, our results demonstrated that Foxp3+ Treg cells in IA patients presented reduced function, which was associated with a defect in Tim-3 upregulation.

Soluble galectin 9 potently enhanced regulatory T-cell formation, a pathway impaired in patients with intracranial aneurysm.

Patients with intracranial aneurysm (IA) present a dysregulated immune system with lower frequency of regulatory T (Treg) cells. Here, we examined whether galectin 9 (Gal-9), the natural ligand of Tim-3, could promote Treg cells in IA patients. We first discovered that the intracellular and extracellular Gal-9 was primarily expressed by CD4+ CD25- T conventional (Tconv) cells, and also by monocytes at lower levels, but rarely by CD4+ CD25+ Treg cells. In IA patients, the Gal-9 expression was significantly lower than in healthy controls. CD4+ CD25- Tconv cells could be induced into Foxp3-expressing induced Treg (iTreg) cells using a TGF-ß-containing milieu. We found that soluble Gal-9 significantly enhanced this process by potently upregulating the expression of Foxp3, IL-10 and TGF-ß in a concentration-dependent manner. In addition, in the absence of additional Gal-9, the level of Foxp3 upregulation was directly correlated with the level of intrinsic Gal-9 expression. Notably, the strength of external Gal-9-mediated effects was significantly lower in IA patients than in healthy controls. Using a Tim-3 blocking antibody, we found that the promotion of iTreg development by soluble Gal-9 was dependent on the Tim-3 signalling pathway. Overall, our investigations demonstrated that Gal-9 presented a critical role in the development of iTreg cells. However, this mechanism was impaired in IA patients due to lower expression of both Gal-9 and Tim-3.

NF-κB-Mediated Inflammation in the Pathogenesis of Intracranial Aneurysm and Subarachnoid Hemorrhage. Does Autophagy Play a Role?

The rupture of saccular intracranial aneurysms (IA) is the commonest cause of non-traumatic subarachnoid hemorrhage (SAH)—the most serious form of stroke with a high mortality rate. Aneurysm walls are usually characterized by an active inflammatory response, and NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) has been identified as the main transcription factor regulating the induction of inflammation-related genes in IA lesions. This transcription factor has also been related to IA rupture and resulting SAH. We and others have shown that autophagy interacts with inflammation in many diseases, but there is no information of such interplay in IA. Moreover, NF-κB, which is a pivotal factor controlling inflammation, is regulated by autophagy-related proteins, and autophagy is regulated by NF-κB signaling. It was also shown that autophagy mediates the normal functioning of vessels, so its disturbance can be associated with vessel-related disorders. Early brain injury, delayed brain injury, and associated cerebral vasospasm are among the most serious consequences of IA rupture and are associated with impaired function of the autophagy⁻lysosomal system. Further studies on the role of the interplay between autophagy and NF-κB-mediated inflammation in IA can help to better understand IA pathogenesis and to identify IA patients with an increased SAH risk.