CXC Ligand Research Articles

CXCL5 impedes CD8+ T cell immunity by upregulating PD-L1 expression in lung cancer via PXN/AKT signaling phosphorylation and neutrophil chemotaxis

BackgroundLung cancer remains one of the most prevalent cancer types worldwide, with a high mortality rate. Upregulation of programmed cell death protein 1 (PD-1) and its ligand (PD-L1) may represent a key mechanism for evading immune surveillance. Immune checkpoint blockade (ICB) antibodies against PD-1 or PD-L1 are therefore widely used to treat patients with lung cancer. However, the mechanisms by which lung cancer and neutrophils in the microenvironment sustain PD-L1 expression and impart stronger inhibition of CD8+ T cell function remain unclear.MethodsWe investigated the role and underlying mechanism by which PD-L1+ lung cancer and PD-L1+ neutrophils impede the function of CD8+ T cells through magnetic bead cell sorting, quantitative real-time polymerase chain reaction (RT-PCR), western blotting, enzyme-linked immunosorbent assays, confocal immunofluorescence, gene silencing, flow cytometry, etc. In vivo efficacy and safety studies were conducted using (Non-obeseDiabetes/severe combined immune deficiency) SCID/NOD mice. Additionally, we collected clinical and prognostic data from 208 patients who underwent curative lung cancer resection between 2017 and 2018.ResultsWe demonstrated that C-X-C motif chemokine ligand 5 (CXCL5) is markedly overexpressed in lung cancer cells and is positively correlated with a poor prognosis in patients with lung cancer. Mechanistically, CXCL5 activates the phosphorylation of the Paxillin/AKT signaling cascade, leading to upregulation of PD-L1 expression and the formation of a positive feedback loop. Moreover, CXCL5 attracts neutrophils, compromising CD8+ T cell-dependent antitumor immunity. These PD-L1+ neutrophils aggravate CD8+ T cell exhaustion following lung cancer domestication. Combined treatment with anti-CXCL5 and anti-PD-L1 antibodies significantly inhibits tumor growth in vivo.ConclusionsOur findings collectively demonstrate that CXCL5 promotes immune escape through PD-L1 upregulation in lung cancer and neutrophils chemotaxis through autocrine and paracrine mechanisms. CXCL5 may serve as a potential therapeutic target in synergy with ICBs in lung cancer immunotherapy.

IL-17 is associated with disease severity and targetable inflammatory processes in heart failure.

Heart failure (HF) is recognized as an inflammatory disease in which cytokines play an important role. In animal HF models, interleukin-17A (IL-17) has been linked to deterioration of cardiac function and fibrosis, whereas knock-out of IL-17 showed beneficial cardiac effects. However, there is limited evidence of IL-17 involvement in patients with HF. This study aims to investigate the clinical characteristics, outcomes, and pathophysiological processes associated with circulating IL-17 concentrations in patients with HF. IL-17 was measured by ELISA in 2082 patients diagnosed with HF along with 363 circulating proteins using proximity extension assay technology for differential expression and pathway analysis. Data were validated in an independent cohort of 1737 patients with HF. Patients with elevated IL-17 concentrations had more severe HF, as reflected by more frequent current or previous hospitalizations for HF, higher New York Heart Association functional class (NYHA) and higher levels of N-terminal pro-B-type natriuretic peptide (NT-proBNP). High IL-17 concentrations were independently associated with an increased risk of hospitalization for HF and mortality. In both cohorts, the most strongly up-regulated proteins in patients with high IL-17 were fibroblast growth factor 21 (FGF-21), interleukin-6 (IL-6), C-X-C motif chemokine ligand 13 (CXCL13), tumour necrosis factor receptor superfamily member 6B (TNFRSF6B) and interleukin-1 receptor antagonist (IL-1RA). Pathway over-representation analysis showed increased activity of pathways related to lymphocyte-mediated immunity, leukocyte activation and regulation of the immune response. In patients with HF, elevated IL-17 concentrations indicate more severe HF and increased activity of inflammatory processes known to be involved in the pathophysiology of HF. IL-17 might hold potential for identifying and targeting inflammation in HF.

Effects of rice fermentation and its bioactive components on UVA-induced oxidative stress and senescence in dermal fibroblasts.

Photoaging, caused by ultraviolet (UV) radiation, is characterized by the senescence of skin cells and reduction of collagens. Although rice fermentation is widely used in the cosmetics, its impact on skin photoaging is still not well understood. Herein, we investigated the possible effectiveness of Maifuyin, a fermented rice product, and its components, succinic acid (SA), and choline, for safeguarding UVA-exposed human dermal fibroblasts (HDFs) against photoaging. In this study, the effects of Maifuyin, SA, and choline on UVA-induced cell death and senescence in fibroblasts were evaluated in cell counting kit-8 (CCK-8), expression of β-galactosidase (β-GAL), and matrix metalloproteinases (MMP)-1. To identify oxidative stress, the investigation focused on reactive oxygen species, glutathione, superoxide dismutase, and malondialdehyde. Additionally, a mRNA sequencing technology (RNA-seq) was applied to study the underlying mechanisms of these components on UVA-induced photoaging. Meanwhile, the level of C-X-C motif chemokine ligand 2 (CXCL2) in the cell supernatant was confirmed to assess the autocrine chemokine level. To reassess the involvement of CXCL2, the expression of β-GAL was evaluated in fibroblasts treated with or without CXCL2. The results indicated that 1 mg/mL Maifuyin and SA inhibited UVA-induced senescence in fibroblasts, MMP-1 expression, and oxidative damage. The RNA-seq revealed 1 mg/mL Maifuyin and SA might be recruited chemokine CXCLs to inhibit MMPs production and fibroblast senescence via TNFα, MAPK, and NF-κB pathways. ELISA results showed a significant reduction of autocrine CXCL2 in UVA-irradiated HDFs by pretreating Maifuyin and SA. The β-GAL staining assay revealed that CXCL2 treatment increased β-GAL activity, while the administration of Maifuyin and SA counteracted this effect in HDFs. These results highlighted the potential use of Maifuyin and SA as promising candidates for anti-photoaging applications.

Integrating UHPLC-Q-TOF-MS/MS, network pharmacology, bioinformatics and experimental validation to uncover the anti-cancer mechanisms of TiaoPi AnChang decoction in colorectal cancer

Ethnopharmacological relevanceThe TiaoPi AnChang Decoction (TPACD), a Traditional Chinese Medicine (TCM) prescription based on Xiangsha Liujunzi Decoction, has demonstrated clinical efficacy as an adjuvant therapy for colorectal cancer (CRC) patients. However, its specific ingredients and potential mechanisms of action remain unclear. Aim of the studyTo identify the primary active ingredients of TPACD, their molecular targets, and potential mechanisms underlying the efficacy of TPACD in CRC treatment. Materials and methodsThis study investigated the clinically validated TCM formula TPACD. In vitro and in vivo experiments were used to demonstrate TPACD's regulatory effects on various malignant phenotypes of tumors, providing basic research support for its anti-cancer activity. To understand its pharmacodynamic basis, we utilized ultra-high performance liquid chromatography-quadrupole-time-of-flight-mass spectrometry/mass spectrometry (UHPLC-Q-TOF-MS/MS) to analyze TPACD constituents present in the bloodstream. Network pharmacology and bioinformatics analyses were used to identify potential active components and their molecular targets for TPACD's therapeutic effects in CRC. Subsequent experiments further elucidated its pharmacological mechanism. ResultsTPACD inhibits various malignant cellular processes, such as cell proliferation, apoptosis, migration, and invasion, and has shown potential anti-CRC activities both in vitro and in vivo. Following the identification of 109 constituents absorbed into the blood from TPACD, network pharmacology analysis predicted 42 potential anti-CRC targets. Clinical analyses highlighted three genes as prognostic key genes of TPACD's therapeutic action: C-X-C motif chemokine ligand 8 (CXCL8), fatty acid binding protein 4 (FABP4), and matrix metallopeptidase 3 (MMP3). Drug sensitivity analyses, molecular docking simulations and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) identified MMP3 as the most promising target for TPACD's anti-CRC action. Enzyme activity assays confirmed that TPACD inhibits MMP3 enzyme activity. Surface plasmon resonance (SPR) characterized the binding affinity between MMP3 and effective active components of TPACD, including luteolin, quercetin, kaempferol, and liensinine. ConclusionsTPACD exhibits anti-CRC activity in vitro and in vivo, with MMP3 identified as a critical target. The active compounds, including luteolin, quercetin, kaempferol, and liensinine, absorbed into the bloodstream, contribute to TPACD's efficacy by targeting MMP3.

Chemokine CXCL9, a marker of inflammaging, is associated with changes of muscle strength and mortality in older men

SummaryOur study examined associations of the CXC motif chemokine ligand 9 (CXCL9), a pro-inflammatory protein implicated in age-related inflammation, with musculoskeletal function in elderly men. We found in certain outcomes both cross-sectional and longitudinal significant associations of CXCL9 with poorer musculoskeletal function and increased mortality in older men. This requires further investigation.PurposeWe aim to determine the relationship of (CXCL9), a pro-inflammatory protein implicated in age-related inflammation, with both cross-sectional and longitudinal musculoskeletal outcomes and mortality in older men.MethodsA random sample from the Osteoporotic Fractures in Men (MrOS) Study cohort (N = 300) was chosen for study subjects that had attended the third and fourth clinic visits, and data was available for major musculoskeletal outcomes (6 m walking speed, chair stands), hip bone mineral density (BMD), major osteoporotic fracture, mortality, and serum inflammatory markers. Serum levels of CXCL9 were measured by ELISA, and the associations with musculoskeletal outcomes were assessed by linear regression and fractures and mortality with Cox proportional hazards models.ResultsThe mean CXCL9 level of study participants (79.1 ± 5.3 years) was 196.9 ± 135.2 pg/ml. There were significant differences for 6 m walking speed, chair stands, physical activity scores, and history of falls in the past year across the quartiles of CXCL9. However, higher CXCL9 was only significantly associated with changes in chair stands (β = − 1.098, p < 0.001) even after adjustment for multiple covariates. No significant associations were observed between CXCL9 and major osteoporotic fracture or hip BMD changes. The risk of mortality increased with increasing CXCL9 (hazard ratio quartile (Q)4 vs Q1 1.98, 95% confidence interval 1.25–3.14; p for trend < 0.001).ConclusionsGreater serum levels of CXCL9 were significantly associated with a decline in chair stands and increased mortality. Additional studies with a larger sample size are needed to confirm our findings.

Senescent endothelial cells promote liver metastasis of uveal melanoma in single-cell resolution

BackgroundUveal melanoma (UM), the most common adult intraocular tumor, is characterized by high malignancy and poor prognosis in advanced stages. Angiogenesis is critical for UM development, however, not only the role of vascular endothelial dysfunction in UM remains unknown, but also their analysis at the single-cell level has been lacking. A comprehensive analysis is essential to clarify the role of the endothelium in the development of UM.MethodsBy using single-cell RNA transcriptomics data of 11 cases of primary and liver metastasis UM, we analyzed the endothelial cell status. In addition, we analyzed and validated ECs in the in vitro model and collected clinical specimens. Subsequently, we explored the impact of endothelial dysfunction on UM cell migration and explored the mechanisms responsible for the endothelial cell abnormalities and the reasons for their peripheral effects.ResultsUM metastasis has a significantly higher percentage of vascular endothelial cells compared to in situ tumors, and endothelial cells in metastasis show significant senescence. Senescent endothelial cells in metastatic tumors showed significant Krüppel-like factor 4 (KLF4) upregulation, overexpression of KLF4 in normal endothelial cells induced senescence, and knockdown of KLF4 in senescent endothelium inhibited senescence, suggesting that KLF4 is a driver gene for endothelial senescence. KLF4-induced endothelial senescence drove tumor cell migration through a senescence-associated secretory phenotype (SASP), of which the most important component of the effector was CXCL12 (C-X-C motif chemokine ligand 12), and participated in the composition of the immunosuppressive microenvironment.ConclusionThis study provides an undesirable insight of senescent endothelial cells in promoting UM metastasis.

The Role of Pericyte Migration and Osteogenesis in Periodontitis

A ligature-induced periodontitis model was established in wild-type and CD146CreERT2; RosatdTomato mice to explore the function of pericytes in alveolar bone formation. We found that during periodontitis progression and periodontal wound healing, CD146+/NG2+ pericytes were enriched in the periodontal tissue areas, which could migrate to the alveolar bone surface and colocalize with ALP+/OCN+ osteoblasts. Chemokine C-X-C motif receptor 4 (CXCR4) inhibition using AMD3100 blocked CD146-Cre+ pericyte migration and osteogenesis, as well as further exacerbated periodontitis-associated bone loss. Next, primary pericytes were sorted out by magnetic-activated cell sorting and demonstrated that C-X-C motif chemokine ligand 12 (CXCL12) promotes pericyte migration and osteogenesis via CXCL12-CXCR4-Rac1 signaling. Finally, the local administration of an adeno-associated virus for Rac1 overexpression in NG2+ pericytes promotes osteoblast differentiation of pericytes and increases alveolar bone volume in periodontitis. Thus, our results provided the evidence that pericytes may migrate and osteogenesis via the CXCL12-CXCR4-Rac1 axis during the pathological process of periodontitis.

Baohuoside I chemosensitises breast cancer to paclitaxel by suppressing extracellular vesicle/CXCL1 signal released from apoptotic cells.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype and chemotherapy is the cornerstone treatment for TNBC. Regrettably, emerging findings suggest that chemotherapy facilitates pro-metastatic changes in the tumour microenvironment. Extracellular vesicles (EVs) have been highly implicated in cancer drug resistance and metastasis. However, the effects of the EVs released from dying cancer cells on TNBC prognosis and corresponding therapeutic strategies have been poorly investigated. This study demonstrated that paclitaxel chemotherapy elicited CXCL1-enriched EVs from apoptotic TNBC cells (EV-Apo). EV-Apo promoted the chemoresistance and invasion of co-cultured TNBC cells by polarizing M2 macrophages through activating PD-L1 signalling. However, baohuoside I (BHS) remarkably sensitized the co-cultured TNBC cells to paclitaxel chemotherapy via modulating EV-Apo signalling. Mechanistically, BHS remarkably decreased C-X-C motif chemokine ligand 1 (CXCL1) cargo within EV-Apo and therefore attenuated macrophage M2 polarization by suppressing PD-L1 activation. Additionally, BHS decreased EV-Apo release by diminishing the biogenesis of intraluminal vesicles (ILVs) within multivesicular bodies (MVBs) of TNBC cells. Furthermore, BHS bound to the LEU104 residue of flotillin 2 (FLOT2) and interrupted its interaction with RAS oncogene family member 31 (RAB31), leading to the blockage of RAB31-FLOT2 complex-driven ILV biogenesis. Importantly, BHS remarkably chemosensitised paclitaxel to inhibit TNBC metastasis in vivo by suppressing EV-ApoCXCL1-induced PD-L1 activation and M2 polarization of tumour-associated macrophages (TAMs). This pioneering study sheds light on EV-ApoCXCL1 as a novel therapeutic target to chemosensitise TNBC, and presents BHS as a promising chemotherapy adjuvant to improve TNBC chemosensitivity and prognosis by disturbing EV-ApoCXCL1 biogenesis.

LncRNA lnc-SPRR2G-2 contributes to keratinocyte hyperproliferation and inflammation in psoriasis by activating the STAT3 pathway and downregulating KHSRP

Psoriasis is a chronic inflammatory disease characterized by increased keratinocyte proliferation and local inflammation. Long noncoding RNAs (lncRNAs) play important regulatory roles in many immune-mediated diseases, including psoriasis. In this study, we aimed to investigate the role and mechanism of lnc-SPRR2G-2 (SPRR2G) in M5-treated psoriatic keratinocytes.Fluorescence in situ hybridization and quantitative real-time polymerase chain reaction (qRT-PCR) showed that lnc-SPRR2G-2 was significantly upregulated in psoriasis tissues and psoriatic keratinocytes. In psoriatic keratinocytes, functional and molecular experiment analyses demonstrated that SPRR2G regulated proliferation, cell cycle and apoptosis, and induced the expression of S100 calcium binding protein A7 (S100A7), interleukin (IL)-1β, IL-8 and C-X-C motif chemokine ligand 10 (CXCL10). The function of SPRR2G in psoriasis is related to the STAT3 signaling pathway and can be inhibited by a STAT3 inhibitor. Moreover, KH-type splicing regulatory protein (KHSRP) was proved to be regulated by lnc-SPRR2G-2 and to control the mRNA decay of psoriasis-related cytokines (p < 0.05). In summary, we reported the functions of lnc-SPRR2G-2 and KHSRP in psoriasis. Our findings provide new insights for the further exploration of the pathogenesis and treatment of psoriasis.

Vagus nerve modulates acute-on-chronic liver failure progression via CXCL9

Abstract Background: Hepatic inflammatory cell accumulation and the subsequent systematic inflammation drive acute-on-chronic liver failure (ACLF) development. Previous studies showed that the vagus nerve exerts anti-inflammatory activity in many inflammatory diseases. Here, we aimed to identify the key molecule mediating the inflammatory process in ACLF and reveal the neuroimmune communication arising from the vagus nerve and immunological disorders of ACLF. Methods: Proteomic analysis was performed and validated in ACLF model mice or patients, and intervention animal experiments were conducted using neutralizing antibodies. PNU-282987 (acetylcholine receptor agonist) and vagotomy were applied for perturbing vagus nerve activity. Single-cell RNA sequencing (scRNA-seq), flow cytometry, immunohistochemical and immunofluorescence staining, and CRISPR/Cas9 technology were used for in vivo or in vitro mechanistic studies. Results: The unbiased proteomics identified C-X-C motif chemokine ligand 9 (CXCL9) as the greatest differential protein in the livers of mice with ACLF and its relation to the systematic inflammation and mortality were confirmed in patients with ACLF. Interventions on CXCL9 and its receptor C-X-C chemokine receptor 3 (CXCR3) improved liver injury and decreased mortality of ACLF mice, which were related to the suppressing of hepatic immune cells’ accumulation and activation. Vagus nerve stimulation attenuated while vagotomy aggravated the expression of CXCL9 and the severity of ACLF. Blocking CXCL9 and CXCR3 ameliorated liver inflammation and increased ACLF-associated mortality in ACLF mice with vagotomy. scRNA-seq revealed that hepatic macrophages served as the major source of CXCL9 in ACLF and were validated by immunofluorescence staining and flow cytometry analysis. Notably, the expression of CXCL9 in macrophages was modulated by vagus nerve-mediated cholinergic signaling. Conclusions: Our novel findings highlighted that the neuroimmune communication of the vagus nerve–macrophage–CXCL9 axis contributed to ACLF development. These results provided evidence for neuromodulation as a promising approach for preventing and treating ACLF.

Silencing CXCL16 alleviate neuroinflammation and M1 microglial polarization in mouse brain hemorrhage model and BV2 cell model through PI3K/AKT pathway.

Neuroinflammation and microglia polarization play pivotal roles in brain injury induced by intracerebral hemorrhage (ICH). Despite the well-established involvement of CXC motif chemokine ligand 16 (CXCL16) in regulating inflammatory responses across various diseases, its specific functions in the context of neuroinflammation and microglial polarization following ICH remain elusive. In this study, we investigated the impact of CXCL16 on neuroinflammation and microglia polarization using both mouse and cell models. Our findings revealed elevated CXCL16 expression in mice following ICH and in BV2 cells after lipopolysaccharide (LPS) stimulation. Specific silencing of CXCL16 using siRNA led to a reduction in the expression of neuroinflammatory factors, including IL-1β and IL-6, as well as decreased expression of the M1 microglia marker iNOS. Simultaneously, it enhanced the expression of anti-inflammatory factors such as IL-10 and the M2 microglia marker Arg-1. These results were consistent across both mouse and cell models. Intriguingly, co-administration of the PI3K-specific agonist 740 Y-P with siRNA in LPS-stimulated cells reversed the effects of siRNA. In conclusion, silencing CXCL16 can positively alleviate neuroinflammation and M1 microglial polarization in BV2 inflammation models and ICH mice. Furthermore, in BV2 cells, this beneficial effect is mediated through the PI3K/Akt pathway. Inhibition of CXCL16 could be a novel approach for treating and diagnosing cerebral hemorrhage.

Asperuloside inhibits the activation of pancreatic cancer-associated fibroblasts via activating transcription factor 6

BackgroundPancreatic cancer-associated fibroblasts (CAFs) play a crucial role in tumor progression and immune evasion. Asperuloside (ASP) is an iridoid glycoside with potential anti-tumor properties. This study aimed to explore the molecular mechanisms of ASP on CAFs, particularly focusing on its effects on activating transcription factor 6 (ATF6), a key regulator of endoplasmic reticulum stress.MethodCAFs were treated with different concentrations of ASP (0, 1, 3, and 5 mM), and the role of ATF6 was investigated by over-expressing it in CAFs. Subsequently, western blot was used to detect ATF6, α-smooth muscle actin (α-SMA), fibroblast activating protein (FAP), and vimentin protein levels in CAFs. The collagen gel contraction assay and Transwell assay were applied to evaluate the contraction and migration ability of CAFs. In addition, the interleukin (IL)-6, C–C motif chemokine ligand (CCL)-2, and C-X-C motif chemokine ligand (CXCL)-10 levels were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR).ResultsCAFs had significantly higher expression levels of α-SMA, FAP, and vimentin compared to normal fibroblasts (NFs). ASP significantly inhibited the activation, contraction, and migration of CAFs in a concentration-dependent manner. ASP treatment also reduced the expression of cytokines (IL-6, CCL2, and CXCL10) and down-regulated ATF6 levels. Over-expression of ATF6 mitigated the inhibitory effects of ASP.ConclusionASP exerts its anti-tumor effects by down-regulating ATF6, thereby inhibiting the activation and function of pancreatic CAFs. These findings suggest that ASP could be a promising therapeutic agent for pancreatic cancer by modulating the tumor microenvironment.

Repeated Infusions of Bone-Marrow-Derived Mesenchymal Stem Cells over 8 Weeks for Steroid-Refractory Chronic Graft-versus-Host Disease: A Prospective, Phase I/II Clinical Study.

Chronic graft-versus-host disease (cGVHD) is a long-term complication of allogeneic hematopoietic stem cell transplantation associated with poor quality of life and increased morbidity and mortality. Currently, there are several approved treatments for patients who do not respond to steroids, such as ruxolitinib. Nevertheless, a significant proportion of patients fail second-line treatment, indicating the need for novel approaches. Mesenchymal stem cells (MSCs) have been considered a potential treatment approach for steroid-refractory cGVHD. To evaluate the safety and efficacy of repeated infusions of MSCs, we administered intravenous MSCs every two weeks to ten patients with severe steroid-refractory cGVHD in a prospective phase I clinical trial. Each patient received a total of four doses, with each dose containing 1 × 106 cells/kg body weight from the same donor and same passage. Patients were assessed for their response to treatment using the 2014 National Institutes of Health (NIH) response criteria during each visit. Ten patients with diverse organ involvement were enrolled, collectively undergoing 40 infusions as planned. Remarkably, the MSC infusions were well tolerated without severe adverse events. Eight weeks after the initial MSC infusion, all ten patients showed partial responses characterized by the amelioration of clinical symptoms and enhancement of their quality of life. The overall response rate was 60%, with a complete response rate of 20% and a partial response (PR) rate of 40% at the last follow-up. Overall survival was 80%, with a median follow-up of 381 days. Two patients died due to relapse of their primary disease. Immunological analyses revealed a reduction in inflammatory markers, including Suppression of Tumorigenicity 2 (ST2), C-X-C motif chemokine ligand (CXCL)10, and Secreted phosphoprotein 1(SPP1), following the MSC treatment. Repeated MSC infusions proved to be both feasible and safe, and they may be an effective salvage therapy in patients with steroid-refractory cGVHD. Further large-scale clinical studies with long-term follow-up are needed in the future to determine the role of MSCs in cGVHD.

Quantifying spatial CXCL9 distribution with image analysis predicts improved prognosis of triple-negative breast cancer.

Background: The C-X-C motif chemokine ligand 9 (CXCL9) plays a pivotal role in tumor immunity by recruiting and activating immune cells. However, the relationship between CXCL9 expression and prognosis in triple-negative breast cancer (TNBC) is unclear. Methods: We investigated CXCL9 mRNA expression, clinicopathological features, and prognosis in TNBC patients. We also used computational image analysis to quantify and assess the distribution of CXCL9 protein in the tumor core (TC) and invasive margin (IM). Results: CXCL9 mRNA expression was significantly higher in TNBC tumors compared to normal tissue (p < 0.001) and was associated with smaller tumors (p = 0.022) and earlier stages (p = 0.033). High CXCL9 mRNA expression was correlated with improved overall survival (OS) in three independent cohorts (all p < 0.05). In a separate analysis, low CXCL9 protein expression was associated with increased lymph node metastasis (p = 0.018 and p = 0.036). High CXCL9 protein expression in the TC, IM, or both was associated with prolonged OS (all p < 0.001). Conclusion: High CXCL9 expression, at both the mRNA and protein levels, is associated with improved prognosis in TNBC patients. CXCL9 expression in the TC and/or IM may be an independent prognostic factor.

HYDROALCOHOLIC EXTRACT OF Lepidium draba L. AMELIORATES CAPECITABINE -INDUCED ENTEROCOLITIS IN RATS

This study investigated the protective properties of Lepidium draba L. hydroalcoholic extract (LDHE) against enterocolitis induced by Capecitabine (CT), utilizing biochemical, molecular, and histopathological analyses. A study was conducted involving 50 Wistar rats divided into 5 groups of ten rats over 60 days: healthy, 400 mg/kg LDHE, 20 mg/kg CT, and two co-treatment groups receiving both CT and 200 and 400 mg/kg LDHE groups. On the 61st day, serum nitric oxide, antidiuretic hormone (ADH), arginine vasopressin (AVP), tumor necrosis factor-α, interleukin-6, chemokine C-X-C motif ligand 1 (CXCL-1), and interleukin-1β levels were measured, along with the activity of glutathione peroxidase, catalase, and superoxide dismutase enzymes. To evaluate tissue oxidative stress in the intestine, measurements were taken for FRAP, thiol, and TBARS levels. Apoptosis in the intestine was assessed by examining the Bax/Bcl-2, caspase-3, and p53 expression via real-time PCR. Furthermore, real-time PCR was employed to evaluate water homeostasis by examining the AQP3, AQP8, and AQP10 expression, while protein expression was analyzed using western blotting. LDHE extract effectively regulates inflammatory cytokine levels and modulates ADH and AVP levels, thereby preserving serum and intestinal osmotic balance. Furthermore, it attenuated the Bax/Bcl-2, caspase-3, and p53 mitochondrial apoptotic pathways while enhancing the expression of AQP3, AQP8, and AQP10 genes in intestinal tissue. The study suggests that LDHE holds promise in the treatment of enterocolitis in chemotherapy patients. Keywords: Lepidium draba L., Capecitabine, Intestine, Enterocolitis, Apoptosis, Aquaporins

Modeling of Blood-Brain Barrier (BBB) Dysfunction and Immune Cell Migration Using Human BBB-on-a-Chip for Drug Discovery Research.

Blood-brain barrier (BBB) dysfunction is a key feature in neuroimmunological and neurodegenerative diseases. In this study, we developed a microfluidic human BBB-on-a-chip to model barrier dysfunction and immune cell migration using immortalized TY10 brain endothelial cells, pericytes, and astrocytes. It was found that immortalized TY10 brain endothelial cells developed a microvascular structure under flow. Pericytes were localized on the basal side surrounding the TY10 microvascular structure, showing an in vivo-like structure. Barrier integrity increased under co-culture with pericytes. In addition, both ethylenediaminetetraacetic acid (EDTA) and anti-Claudin-5 (CLDN5) neutralizing antibody caused a decrease in the transendothelial electrical resistance (TEER). EDTA caused the leakage of 20 kDa dextran, suggesting different effects on the BBB based on the mechanism of action, whereas anti-CLDN5 antibody did not cause leakage. In the tri-culture model, human T cells migrated through endothelial vessels towards basal C-X-C motif chemokine ligand 12 (CXCL12). The live-imaging analysis confirmed the extravasation of fluorescence-labelled T cells in a CXCL12-concentration- and time-dependent manner. Our BBB model had an in vivo-like structure and successfully represented barrier dysfunction and transendothelial T cell migration. In addition, our study suggests that the inhibition of CLDN5 attenuates the BBB in humans. This platform has various potential uses in relation to the BBB in both drug discovery research and in elucidating the mechanisms of central nervous system diseases.

Innate lymphoid cell-based immunomodulatory hydrogel microspheres containing Cutibacterium acnes extracellular vesicles for the treatment of psoriasis

Psoriasis is a chronic skin inflammation influenced by dysregulated skin microbiota, with the role of microbiota in psoriasis gaining increasing prominence. Bacterial extracellular vesicles (bEVs) serve as crucial regulators in the interaction between hosts and microbiota. However, the mechanism underlying the therapeutic potential of bEVs from commensal bacteria in psoriasis remains unclear. Here, we investigated the therapeutic role of Cutibacterium acnes (C. acnes)-derived extracellular vesicles (CA-EVs) in psoriasis treatment. To prolong the active duration of CA-EVs, we encapsulated them in gelatin methacrylate (GelMA) to fabricate hydrogel microspheres (CA-EVs@GHM) with sustained release properties. As GelMA degraded, CA-EVs were gradually released, maintaining a high concentration in mouse skin even 96 h post-treatment. In human keratinocyte cells (HaCaT), CA-EVs@GHM enhanced resistance to Staphylococcus aureus (S. aureus), promoted proliferation and migration of HaCaT cells exposed to S. aureus, and significantly reduced the expression of inflammatory genes such as interleukin (IL)-6 and C-X-C motif chemokine ligand 8 (CXCL8). In vivo, CA-EVs@GHM, more potent than CA-EVs alone, markedly attenuated proinflammatory gene expression, including tumor necrosis factor (TNF), Il6, Il17a, Il22 and Il23a in imiquimod (IMQ)-induced psoriasis-like mice, and restored skin barrier function. 16S rRNA sequencing revealed that CA-EVs@GHM might provide therapeutic effects against psoriasis by restoring microbiota diversity on the back skin of mice, reducing Staphylococcus colonization, and augmenting lipid metabolism. Furthermore, flow cytometry analysis showed that CA-EVs@GHM prevented the conversion of type 2 innate lymphoid cells (ILC2) to type 3 innate lymphoid cells (ILC3) in psoriasis-like mouse skin, reducing the pathogenic ILC3 population and suppressing the secretion of IL-17 and IL-22. In summary, our findings demonstrate that the long-term sustained release of CA-EVs alleviated psoriasis symptoms by controlling the transformation of innate lymphoid cells (ILCs) subgroups and restoring skin microbiota homeostasis, thus offering a promising therapy for psoriasis treatment. Statement of significanceCutibacterium acnes, which is reduced in psoriasis skin, has been reported to promote skin homeostasis by regulating immune balance. Compared to live bacteria, bacterial extracellular vesicles (bEVs) are less prone to toxicity and safety concerns. bEVs play a pivotal role in maintaining bacterial homeostasis and modulating the immune system.However,bEVs without sustained release materials are unable to function continuously in chronic diseases. Therefore, we utilized hydrogel microspheres to encapsulate Cutibacterium acnes (C. acnes)-derived extracellular vesicles (CA-EVs), enabling long term sustained release. Our findings indicate that, CA-EVs loaded gelatin methacrylate hydrogel microspheres (CA-EVs@GHM) showed superior therapeutic effects in treating psoriasis compared to CA-EVs. CA-EVs@GHM exhibited a more significant regulation of pathological type 3 innate lymphoid cells (ILC3) and skin microbiota, providing a promising approach for microbiota-derived extracellular vesicle therapy in the treatment of skin inflammation.

Scutellarein Suppresses the Production of ROS and Inflammatory Mediators of LPS-Activated Bronchial Epithelial Cells and Attenuates Acute Lung Injury in Mice.

Scutellarein is a key active constituent present in many plants, especially in Scutellaria baicalensis Georgi and Erigeron breviscapus (vant.) Hand-Mazz which possesses both anti-inflammatory and anti-oxidative activities. It also is the metabolite of scutellarin, with the ability to relieve LPS-induced acute lung injury (ALI), strongly suggesting that scutellarein could suppress respiratory inflammation. The present study aimed to investigate the effects of scutellarein on lung inflammation by using LPS-activated BEAS-2B cells (a human bronchial epithelial cell line) and LPS-induced ALI mice. The results showed that scutellarein could reduce intracellular reactive oxygen species (ROS) accumulation through inhibiting the activation of NADPH oxidases, markedly downregulating the transcription and translation of pro-inflammatory cytokines, including interleukin-6 (IL-6), C-C motif chemokine ligand 2 (CCL2), and C-X-C motif chemokine ligand (CXCL) 8 in LPS-activated BEAS-2B cells. The mechanism study revealed that it suppressed the phosphorylation and degradation of IκBα, consequently hindering the translocation of p65 from the cytoplasm to the nucleus and its subsequent binding to DNA, thereby decreasing NF-κB-regulated gene transcription. Notably, scutellarein had no impact on the activation of AP-1 signaling. In LPS-induced ALI mice, scutellarein significantly decreased IL-6, CCL2, and tumor necrosis factor-α (TNF-α) levels in the bronchoalveolar lavage fluid, attenuated lung injury, and inhibited neutrophil infiltration. Our findings suggest that scutellarein may be a beneficial agent for the treatment of infectious pneumonia by virtue of its anti-oxidative and anti-inflammatory activities.

IL-17A-induced cancer-associated fibroblasts releases CXCL12 to promote lung adenocarcinoma progression via Wnt/β-Catenin signaling pathway

BackgroundCancer-associated fibroblasts (CAFs) and their secretion, C-X-C motif chemokine ligand 12 (CXCL12), play an important role in the development of lung adenocarcinoma (LUAD). Interleukin 17A (IL-17A) is also crucial in regulating tumor progression. Herein, we explored the specific relationships between these two factors and their mechanisms in the progression of LUAD. MethodsImmunohistochemistry was utilized to assess the differential expression levels of IL-17A and CXCL12 in tumor versus normal tissues of LUAD patients, followed by gene correlation analysis. Cell counting kit-8 (CCK8), wound-healing and transwell assays were performed to investigate the effect of IL-17A on the function of LUAD cells. qPCR, immunofluorescence, immunohistochemistry and western blot analyses were conducted to elucidate the potential mechanism by which IL-17A facilitates the development of LUAD via CXCL12. Male BALB-C nude mice were used to explore the role of IL-17A in subcutaneous LUAD mouse models. ResultsElevated expression levels of IL-17A and CXCL12 were observed in LUAD tissues, exhibiting a positive correlation. Further studies revealed that IL-17A could stimulate CAFs to enhance the release of CXCL12, thereby facilitating the growth, proliferation, and metastasis of LUAD. The binding of CXCL12 to its specific receptor influences the activation of the Wnt/β-Catenin pathway, which in turn affects the progression of LUAD. In vivo experiments have demonstrated that IL-17A enhances the growth of LUAD tumors by facilitating the secretion of CXCL12. Conversely, inhibiting CXCL12 has been demonstrated to impede tumor growth. ConclusionsWe discovered that IL-17A promotes the release of CAFs-derived CXCL12, which in turn facilitates the development of LUAD via the Wnt/β-Catenin signaling pathway.

Predicting Major Adverse Carotid Cerebrovascular Events in Patients with Carotid Stenosis: Integrating a Panel of Plasma Protein Biomarkers and Clinical Features-A Pilot Study.

Background: Carotid stenosis (CS) is an atherosclerotic disease of the carotid artery that can lead to devastating cardiovascular outcomes such as stroke, disability, and death. The currently available treatment for CS is medical management through risk reduction, including control of hypertension, diabetes, and/or hypercholesterolemia. Surgical interventions are currently suggested for patients with symptomatic disease with stenosis >50%, where patients have suffered from a carotid-related event such as a cerebrovascular accident, or asymptomatic disease with stenosis >60% if the long-term risk of death is <3%. There is a lack of current plasma protein biomarkers available to predict patients at risk of such adverse events. Methods: In this study, we investigated several growth factors and biomarkers of inflammation as potential biomarkers for adverse CS events such as stroke, need for surgical intervention, myocardial infarction, and cardiovascular-related death. In this pilot study, we use a support vector machine (SVM), random forest models, and the following four significantly elevated biomarkers: C-X-C Motif Chemokine Ligand 6 (CXCL6); Interleukin-2 (IL-2); Galectin-9; and angiopoietin-like protein (ANGPTL4). Results: Our SVM model best predicted carotid cerebrovascular events with an area under the curve (AUC) of >0.8 and an accuracy of 0.88, demonstrating strong prognostic capability. Conclusions: Our SVM model may be used for risk stratification of patients with CS to determine those who may benefit from surgical intervention.