Expression Of Chemokines Research Articles

Lactate protects neurons and astrocytes against ischemic injury by modulating Ca2+ homeostasis and inflammatory response.

Lactate is now considered an additional fuel or signaling molecule in the brain. In this study, using an oxygen-glucose deprivation (OGD) model, we found that treatment with lactate inhibited the global increase in intracellular calcium ion concentration ([Ca2+]) in neurons and astrocytes, decreased the percentage of dying cells, and caused a metabolic shift in astrocytes and neurons toward aerobic oxidation of substrates. OGD resulted in proinflammatory changes and increased expression of cytokines and chemokines, whereas incubation with lactate reduced these changes. Pure astrocyte cultures were less sensitive than neuroglia cultures during OGD. Astrocytes exposed to lipopolysaccharide (LPS) also showed pro-inflammatory changes that were reduced by incubation with lactate. Our study suggests that lactate may have neuroprotective effects under ischemic and inflammatory conditions.

Abstract C044: Macropinocytic stress reprograms cancer-associated fibroblasts and enhances therapeutic responses in pancreatic cancer

Abstract Macropinocytosis is a fundamental metabolic pathway that has been demonstrated to support the survival and function of tumor cells and cancer-associated fibroblasts (CAFs) in pancreatic ductal adenocarcinoma (PDAC) through in vitro studies and xenograft mouse models. However, the impact of macropinocytosis on the PDAC tumor microenvironment in an immune-competent setting remains poorly understood. In this study, we utilized the selective inhibitor 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) to block macropinocytosis in both tumor cells and CAFs in syngeneic PDAC mouse models. We observed that EIPA treatment inhibited PDAC tumor growth, which was accompanied by reduced tumor proliferation and increased cell apoptosis. Moreover, EIPA treatment decreased the presence of myofibroblastic CAFs and collagen deposition within PDAC tumors, while also promoting the expansion of tumor vasculature. By modifying the tumor stroma, EIPA priming enhanced drug delivery and improved the effectiveness of PDAC chemotherapy. Interestingly, immune profiling of PDAC tumors revealed that EIPA administration specifically increased the infiltration of CD8+ and CD4+ T cells. We discovered that EIPA induced changes in the cytokine/chemokine landscape within tumors by triggering an inflammatory CAF (iCAF)-like phenotype. Mechanistically, we discerned that inhibition of macropinocytosis triggers metabolic stress that in turn activates stress-responsive transcription factors and promotes the expression of early-phase proinflammatory cytokines and chemokines in mouse CAFs. Notably, combination treatment with EIPA and immune checkpoint blockade (ICB) reduced PDAC tumor burden and significantly inhibited tumor metastasis. Overall, our findings establish that macropinocytosis functions to support the barrier function of the stroma by maintaining fibroblastic identity and provide further evidence that inhibition of macropinocytosis represents a promising therapeutic modality in PDAC. Citation Format: Yijuan Zhang, Swetha Maganti, Jennifer Hope, Li Ling, Cheska Marie Galapate, Florent Carrette, Linda Bradley, Cosimo Commisso. Macropinocytic stress reprograms cancer-associated fibroblasts and enhances therapeutic responses in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C044.

Analysis of Cytokine and Chemokine Level in Tear Film in Keratoconus Patients before and after Corneal Cross-Linking (CXL) Treatment.

Keratoconus (KC) is a degenerative corneal disorder whose aetiology remains unknown. The aim of our study was to analyse the expressions of cytokines and chemokines in KC patients before and after specified time intervals after corneal cross-linking (CXL) treatment to better understand the molecular mechanisms occurring before and after CXL in KC patients process of corneal regeneration.; Tear samples were gathered from 52 participants immediately after the CXL procedure and during the 12-month follow-up period. All patients underwent a detailed ophthalmological examination and tear samples were collected before and after CXL at regular intervals: 1 day before and after the surgery, at the day 7 visit, and at 1, 3, 6, 9, and 12 months after CXL. The control group consisted of 20 healthy people. 10 patients were women (50%) and 10 were men (50%). The mean age was 30 ± 3 years of age. Tear analysis was performed using the Bio-Plex 3D Suspension Array System. Corneal topography parameters measured by Scheimpflug Camera included: keratometry values (Ks, Kf), PI-Apex, PI-Thinnest, Cylinder.; All the 12 months post-op values of the KC patients' topographic measurements were significantly lower than the pre-op. As for the tear cytokine levels comparison between the patient and control groups, cytokine levels of TNF-α, IL-6, and CXCL-10, among others, were detected in lower amounts in the KC group. The pre-op level of IL-6 exhibited a significant increase the day after CXL, whereas comparing the day after the procedure to 12 months after CXL, this showed a significant decrease. Both TNF-α and IL-1 showed a significant decrease compared to the day before and after CXL. We observed significantly higher levels of IL-1β, IL-10, IFN-γ and TNF-α in moderate and severe keratoconus than in mild keratoconus (p < 0.05). We also demonstrated a statistically significant positive correlation between both pre-op and 12 months after CXL TNF-α, IFN-γ, IL-6 and Ks and Kf values (p < 0.05, r > 0); Alterations of inflammatory mediators in tear fluid after CXL link with topographic changes and may contribute to the development and progression of KC.

Differential cytokine and chemokine expression after ablation vs. resection in colorectal cancer liver metastasis

BackgroundSurgical resection remains the main curative treatment for colorectal liver metastases (CRLM). Radiofrequency ablation (RFA) is increasingly employed for small, deep lying or otherwise inoperable lesions. However, RFA can induce pro-tumorigenic effects on residual tumor cells, hereby possibly promoting tumor recurrence. Contrastingly, post-RFA tumor debris as an antigen source can also generate anti-cancer immune responses. Utilizing this, current studies on combining RFA with immune therapy appear promising. Here, in an attempt to shed light on this controversy, cytokines involved in inflammation, (lymph)angiogenesis, immune cell recruitment and tumor cell invasion were investigated post-RFA versus post-resection in CRLM patients. MethodsCytokine and chemokine serum levels pre-operation, 4 h and 24 h post-operation were analyzed in CRLM patients undergoing RFA (n = 8) or partial hepatectomy (n = 9) using Multiplex immunoassays. Statistical analyses were performed between as well as within individual intervention groups. ResultsPost-RFA, significantly increased levels of acute phase proteins SAA1 and S100A8, IL-6, IL-1Ra, MIP3b (CCL19) and MMP9 were observed along with decreases in Fibronectin, MCP-1 (CCL2), and Tie-2. Post-resection, increased levels of PDGFbb, I309 (CCL1), Apelin, MIF, IL-1b and TNFα were seen. All p-values <0.05. ConclusionPro-inflammatory responses mediated by different cytokines were seen after both RFA and resection, possibly influencing residual tumor cells and tumor recurrence. As both ablation and resection trigger inflammation and immune cell recruitment (albeit via distinct mechanisms), these data suggest that further research may explore combining immune therapy with not only RFA but also resection. Key messageAnalysis of patients' serum after radiofrequency ablation versus resection of colorectal liver metastases (CRLM) showed that these interventions trigger inflammation and immune cell recruitment, via different cyto- and chemokine pathways. This suggests a possible future strategy of combining immune therapy with not only ablative techniques but also with resection of CRLM.

Administration of an AAV vector coding for a P2X7-blocking nanobody-based biologic ameliorates colitis in mice

BackgroundThe pro-inflammatory ATP-gated P2X7 receptor is widely expressed by immune and non-immune cells. Nanobodies targeting P2X7, with potentiating or antagonistic effects, have been developed. Adeno-associated virus (AAV)-mediated gene transfer represents an efficient approach to achieve long-term in vivo expression of selected nanobody-based biologics. This approach (AAVnano) was used to validate the relevance of P2X7 as a target in dextran sodium sulfate (DSS)-induced colitis in mice.ResultsMice received an intramuscular injection of AAV vectors coding for potentiating (14D5-dimHLE) or antagonistic (13A7-Fc) nanobody-based biologics targeting P2X7. Long-term modulation of P2X7 activity was evaluated ex vivo from blood samples. Colitis was induced with DSS in mice injected with AAV vectors coding for nanobody-based biologics. Severity of colitis, colon histopathology and expression of chemokines and cytokines were determined to evaluate the impact of P2X7 modulation. A single injection of an AAV vector coding for 13A7-Fc or 14D5-dimHLE efficiently modulated P2X7 function in vivo from day 15 up to day 120 post-injection in a dose-dependent manner. An AAV vector coding for 13A7-Fc significantly ameliorated DSS-induced colitis and significantly reduced immune cell infiltration and expression of chemokines and proinflammatory cytokines in colonic tissue.ConclusionsWe have demonstrated the validity of AAVnano methodology to modulate P2X7 functions in vivo. Applying this methodological approach to a DSS-induced colitis model, we have shown that P2X7 blockade reduces inflammation and disease severity. Hence, this study confirms the importance of P2X7 as a pharmacological target and suggests the use of nanobody-based biologics as potential therapeutics in inflammatory bowel disease.Graphical

Identification and validation of the association of Janus kinase 2 mutations with the response to immune checkpoint inhibitor therapy

BackgroundJanus kinase 2 (JAK2) mutation plays an important role in T cell immunity. However, the effect of JAK2 mutation on immunotherapy is largely uncharacterized.MethodsIn this study, we analyzed the effect of JAK2 mutation on the efficacy and outcomes of immune checkpoint inhibitor (ICI) therapy in the discovery cohort (n = 662) and the verification cohort (n = 1423). Furthermore, we explored the association of JAK2 mutation with the tumor immune microenvironment in a multiomics cohort.ResultsIn the discovery cohort (n = 662), JAK2 mutant-type patients had a better objective response rate (58.8% vs. 26.7%, P = 0.010), durable clinical benefit (64.7% vs. 38.9%, P = 0.043), progression-free survival (hazard ratio [HR] = 0.431, P = 0.015), and overall survival (HR = 0.378, P = 0.025), relative to JAK2 wild-type patients. Moreover, we further verified the prognostic significance of JAK2 mutation in an independent ICI treatment cohort with a larger sample size (n = 1423). In addition, we discovered that the JAK2 mutation was remarkably related to increased immunogenicity, such as a higher TMB, higher expression of costimulatory molecules and stimulation of antigen processing mechanisms. In addition, JAK2 mutation was positively correlated with activated anticancer immunity, such as infiltration of various immune cells and higher expression of chemokines.ConclusionOur study demonstrates that JAK2 mutation is a novel marker that can be used to effectively predict prognosis and response to ICI therapy.

Immunological dimensions of neuroinflammation and microglial activation: exploring innovative immunomodulatory approaches to mitigate neuroinflammatory progression.

The increasing life expectancy has led to a higher incidence of age-related neurodegenerative conditions. Within this framework, neuroinflammation emerges as a significant contributing factor. It involves the activation of microglia and astrocytes, leading to the release of pro-inflammatory cytokines and chemokines and the infiltration of peripheral leukocytes into the central nervous system (CNS). These instances result in neuronal damage and neurodegeneration through activated nucleotide-binding domain and leucine-rich repeat containing (NLR) family pyrin domain containing protein 3 (NLRP3) and nuclear factor kappa B (NF-kB) pathways and decreased nuclear factor erythroid 2-related factor 2 (Nrf2) activity. Due to limited effectiveness regarding the inhibition of neuroinflammatory targets using conventional drugs, there is challenging growth in the search for innovative therapies for alleviating neuroinflammation in CNS diseases or even before their onset. Our results indicate that interventions focusing on Interleukin-Driven Immunomodulation, Chemokine (CXC) Receptor Signaling and Expression, Cold Exposure, and Fibrin-Targeted strategies significantly promise to mitigate neuroinflammatory processes. These approaches demonstrate potential anti-neuroinflammatory effects, addressing conditions such as Multiple Sclerosis, Experimental autoimmune encephalomyelitis, Parkinson's Disease, and Alzheimer's Disease. While the findings are promising, immunomodulatory therapies often face limitations due to Immune-Related Adverse Events. Therefore, the conduction of randomized clinical trials in this matter is mandatory, and will pave the way for a promising future in the development of new medicines with specific therapeutic targets.

Retraction: Association study of inflammatory cytokine and chemokine expression in hand foot and mouth disease.

Retraction: Association study of inflammatory cytokine and chemokine expression in hand foot and mouth disease.

In vitro inflammatory multi-cellular model of osteoarthritis

ObjectiveOsteoarthritis (OA) is a chronic joint disease, with limited treatment options, characterized by inflammation and matrix degradation, and resulting in severe pain or disability. Progressive inflammatory interaction among key cell types, including chondrocytes and macrophages, leads to a cascade of intra- and inter-cellular events which culminate in OA induction. In order to investigate these interactions, we developed a multi-cellular in vitro OA model, to characterize OA progression, and identify and evaluate potential OA therapeutics in response to mediators representing graded levels of inflammatory severity. MethodsWe compared macrophages, chondrocytes and their co-culture responses to “low” Interleukin-1 (IL-1) or “high” IL-1/tumor necrosis factor (IL-1/TNF) levels of inflammation. We also investigated response changes following the administration of dexamethasone (DEX) or mesenchymal stromal cell (MSC) treatment via a combination of gene expression and secretory changes, reflecting not only inflammation, but also chondrocyte function. ResultsInflamed chondrocytes presented an osteoarthritic-like phenotype characterized by high gene expression of pro-inflammatory cytokines and chemokines, up-regulation of ECM degrading proteases, and down-regulation of chondrogenic genes. Our results indicate that while MSC treatment attenuates macrophage inflammation directly, it does not reduce chondrocyte inflammatory responses, unless macrophages are present as well. DEX however, can directly attenuate chondrocyte inflammation. ConclusionsOur results highlight the importance of considering multi-cellular interactions when studying complex systems such as the articular joint. In addition, our approach, using a panel of both inflammatory and chondrocyte functional genes, provides a more comprehensive approach to investigate disease biomarkers, and responses to treatment.

An Immune Gene Expression Risk Score for Distant Metastases after Radiotherapy for Cervical Cancer.

To develop an immune-based gene expression risk score to identify patients with cervical cancer at increased risk of distant metastases (DM). Tumor biopsies were obtained from 81 patients prior to chemoradiotherapy. Whole-transcriptome RNA sequencing was performed (Illumina NextSeq500). Beginning with 4,723 immune-related genes, a 55-gene risk score for DM was derived using Cox modeling and principal component analysis. It was validated in independent cohorts of 274 patients treated at the Norwegian Radium Hospital (NRH) and 206 patients from The Cancer Genome Atlas (TCGA). The risk score was predictive of DM (HR, 2.7; P < 0.0001) and lower cause-specific survival (CSS) by univariate analysis (HR, 2.0; P = 0.0003) and multivariate analysis adjusted for clinical factors (DM HR, 3.0; P < 0.0001; CSS HR, 2.2; P = 0.0004). The risk score predicted DM (HR, 1.4; P = 0.05) and CSS (HR, 1.48; P = 0.013) in the NRH cohort and CSS (HR, 1.4; P = 0.03) in TCGA cohort. Higher risk scores were associated with lower CIBERSORT estimates of tumor-infiltrating immune cells, including CD8 T cells and M1 and M2 macrophages (all P < 0.001). Higher risk scores were associated with lower expression (all P < 0.001) of important chemokines (CXCL12, CXCR4), IFN-regulated genes (IRF1, STAT1, IDO1), and immune checkpoint regulators (PD-1, PD-L1, CTLA-4). The immune metastatic risk score addresses important challenges in the treatment of cervical cancer-identifying patients at high risk of DM after radiotherapy. The findings of this study indicate that high tumor mutational burden and a "cold," immune-excluded tumor microenvironment influence distant metastatic recurrence. Further validation of the risk score is needed.

Histone Deacetylase Inhibitors Directly Modulate T Cell Gene Expression and Signaling and Promote Development of Effector-Exhausted T Cells in Murine Tumors.

Epigenetic regulation plays a crucial role in the development and progression of cancer, including the regulation of antitumor immunity. The reversible nature of epigenetic modifications offers potential therapeutic avenues for cancer treatment. In particular, histone deacetylase (HDAC) inhibitors (HDACis) have been shown to promote antitumor T cell immunity by regulating myeloid cell types, enhancing tumor Ag presentation, and increasing expression of chemokines. HDACis are currently being evaluated to determine whether they can increase the response rate of immune checkpoint inhibitors in cancer patients. Although the potential direct effect of HDACis on T cells likely impacts antitumor immunity, little is known about how HDAC inhibition alters the transcriptomic profile of T cells. In this article, we show that two clinical-stage HDACis profoundly impact gene expression and signaling networks in CD8+ and CD4+ T cells. Specifically, HDACis promoted T cell effector function by enhancing expression of TNF-α and IFN-γ and increasing CD8+ T cell cytotoxicity. Consistently, in a murine tumor model, HDACis led to enrichment of CD8+ T cell subsets with high expression of effector molecules (Prf1, Ifng, Gzmk, and Grmb) but also molecules associated with T cell exhaustion (Tox, Pdcd1, Lag3, and Havcr2). HDACis further generated a tumor microenvironment dominated by myeloid cells with immune suppressive signatures. These results indicate that HDACis directly and favorably augment T cell effector function but also increase their exhaustion signal in the tumor microenvironment, which may add a layer of complexity for achieving clinical benefit in combination with immune checkpoint inhibitors.

Characterization of CD8+ T cells in immune-privileged organs of ZIKV-infected Ifnar1-/- mice.

Zika virus (ZIKV) infection caused neurological complications and male infertility, leading to the accumulation of antigen-specific immune cells in immune-privileged organs (IPOs). Thus, it is important to understand the immunological responses to ZIKV in IPOs. We extensively investigated the ZIKV-specific T cell immunity in IPOs in Ifnar1-/- mice, based on an immunodominant epitope E294-302 tetramer. The distinct kinetics and functions of virus-specific CD8+ T cells infiltrated into different IPOs were characterized, with late elevation in the brain and spinal cord. Single epitope E294-302-specific T cells can account for 20-60% of the total CD8+ T cells in the brain, spinal cord, and testicle and persist for at least 90 days in the brain and spinal cord. The E294-302-specific TCRαβs within the IPOs are featured with the majority of clonotypes utilizing TRAV9N-3 paired with diverse TRBV chains, but with distinct αβ paired clonotypes in 7 and 30 days post-infection. Specific chemokine receptors, Ccr2 and Ccr5, were selectively expressed in the E294-302-specific CD8+ T cells within the brain and testicle, indicating an IPO-oriented migration of virus-specific CD8+ T cells after infection. Overall, this study adds to the understanding of virus-specific CD8+ T cell responses for controlling and clearing ZIKV infection in IPOs.IMPORTANCEThe immune-privileged organs (IPOs), such as the central nervous system and testicles, presented pathogenicity and inflammation after Zika virus (ZIKV) infection with infiltrated CD8+ T cells. Our data show that CD8+ T cells keep up with virus increases and decreases in immune-privileged organs. Furthermore, our study provides the first ex vivo comparative analyses of the composition and diversity related to TCRα/β clonotypes across anatomical sites and ZIKV infection phases. We show that the vast majority of TCRα/β clonotypes in tissues utilize TRAV9N-3 with conservation. Specific chemokine expression, including Ccr2 and Ccr5, was found to be selectively expressed in the E294-302-specific CD8+ T cells within the brain and testicle, indicating an IPO-oriented migration of the virus-specific CD8+ T cells after the infection. Our study adds insights into the anti-viral immunological characterization and chemotaxis mechanism of virus-specific CD8+ T cells after ZIKV infection in different IPOs.

TRIM26 alleviates fatal immunopathology by regulating inflammatory neutrophil infiltration during Candida infection.

Fungal infections have emerged as a major concern among immunocompromised patients, causing approximately 2 million deaths each year worldwide. However, the regulatory mechanisms underlying antifungal immunity remain elusive and require further investigation. The E3 ligase Trim26 belongs to the tripartite motif (Trim) protein family, which is involved in various biological processes, including cell proliferation, antiviral innate immunity, and inflammatory responses. Herein, we report that Trim26 exerts protective antifungal immune functions after fungal infection. Trim26-deficient mice are more susceptible to fungemia than their wild-type counterparts. Mechanistically, Trim26 restricts inflammatory neutrophils infiltration and limits proinflammatory cytokine production, which can attenuate kidney fungal load and renal damage during Candida infection. Trim26-deficient neutrophils showed higher proinflammatory cytokine expression and impaired fungicidal activity. We further demonstrated that excessive neutrophils infiltration in the kidney was because of the increased production of chemokines CXCL1 and CXCL2, which are mainly synthesized in the macrophages or dendritic cells of Trim26-deficient mice after Candida albicans infections. Together, our study findings unraveled the vital role of Trim26 in regulating antifungal immunity through the regulation of inflammatory neutrophils infiltration and proinflammatory cytokine and chemokine expression during candidiasis.

Bioinformatics analysis reveals PIH1D1 as an important prognostic marker in breast cancer

R2TP (Rvb1, Rvb2, Tah1, and Pih1) complex was initially discovered as a Heat shock protein 90 associated multimeric protein complex in yeast and is highly conserved in mammals. In Human R2TP complex is known by different names including RUVBL1 for Rvb1, RuvBL2 for Rvb2, RPAP3 (RNA polymerase II-associated protein 3) for Tah1 and PIH1D1 for Pih1, and known to be a specialized Co-chaperone of Hsp-90 protein. This multimeric-protein complex is involved in the assembly and maturation of several multisubunit complexes including RNA polymerase II, small nucleolar ribonucleoproteins, and complexes containing phosphatidylinositol-3-kinase-like kinases. Nevertheless, evidence in the clinical setting is scanty with respect to the expression of PIH1 domain containing 1 (PIH1D1) and its prognostic values in breast cancer (BRCA). Exploring the function of PIH1D1 in BRCA could reveal its therapeutic and translational potential in BRCA. In the current study, we analyzed PIH1D1 expression levels across cancers with the help of a bioinformatics tool and tumor immune estimation resource, and compared with normal and BRCA tissues using tumor, normal, and metastasis plot. Disease-free and overall survival analysis was done by survival plot, in gene expression profiling interactive analysis web server. Furthermore, histological analysis of PIH1D1 protein expression was done with the help of the Human Protein Atlas Database. Clusters and interaction analysis were done using cell line expression cluster toll in Human Protein Atlas. The result of our study shows that PIH1D1 expression was higher in almost all cancer groups and significantly increased in BRCA. Additionally, we found a positive statistical correlation between PIH1D1 and CD4+ and CD8+ T cell infiltration, which is an indication of the immunotherapeutic potential of PIH1D1. The expression of chemokines and receptors was shown to be significantly positively linked with Chemokine (C-C motif) ligand 14 when PIH1D1 was increased. Furthermore, the UALCAN (University of ALabama at Birmingham CANcer) data analysis results also revealed that PIH1D1 was significantly associated with tumor metastasis, menopausal status, grade, and different stages of patients with BRCA. In conclusion, the current study provides the preliminary proof of concept regarding the biomarker potential of PIH1D1. However, further in vivo and in vitro mechanistic studies in detail are required to explore the therapeutic potential of PIH1D1 in BRCA.

Systemic autoimmune abnormalities alter the morphology of mucosa-associated lymphoid tissues in the rectum of MRL/MpJ-Faslpr/lpr mice.

Systemic autoimmune diseases (ADs) might affect the morphology and function of gut-associated lymphoid tissue (LTs) indirectly; however, their exact relationship remains unclear. Therefore, we investigated mouse LTs in the anorectal canal and morphologically compared them between MRL/MpJ-Fas+/+ and MRL/MpJ-Faslpr/lpr mice. LT aggregations, also known as rectal mucosa-associated lymphoid tissues (RMALTs), were exclusively seen in the lamina propria and submucosa of the rectum. The mean size and number of the LT aggregations both significantly increased in MRL/MpJ-Faslpr/lpr mice compared to those in MRL/MpJ-Fas+/+ mice. The distance from the anorectal junction to the first LT aggregate was significantly shorter in MRL/MpJ-Faslpr/lpr mice than that in MRL/MpJ-Fas+/+ mice. Immunostaining revealed that the RMALTs included CD3+, CD4+, and CD8+ T cells; B220+ B cells; IBA1+ macrophages; Ki67+ proliferative cells; and PNAd+ high-endothelial venules (HEVs). The numbers of macrophages, proliferative cells, CD4+ T cells, CD8+ T cells, and HEVs were significantly increased in MRL/MpJ-Faslpr/lpr mice compared to those in MRL/MpJ mice. Furthermore, the gene expression levels of chemokines (Cxcl9 and Cxcl13) and their corresponding receptors (Cxcr3 and Cxcr5) were significantly higher in MRL/MpJ-Faslpr/lpr mice than those in MRL/MpJ-Fas+/+ mice. Although the morphology of rectal epithelium was comparable between the strains, M cell number was significantly higher in MRL/MpJ-Faslpr/lpr mice than in MRL/MpJ-Fas+/+ mice. Thus, ADs could alter RMALT morphology, and quantitative changes in T-cell subsets, proliferative cells, macrophages, HEVs, chemokine expression, and M cells could affect their cell composition and development.

Identification of the prognostic immune subtype in copy-number high endometrial cancer.

The TCGA molecular subtype of endometrial cancer (EC) is widely applied, among which the copy-number high (CNH) subtype has the poorest prognosis. However, the heterogeneity of this subtype remains elusive. In this study, we aimed to identify heterogeneous immune subtypes in CNH EC and explore their prognostic significance. We collected 60 CNH EC cases in the TCGA database and performed unsupervised cluster analysis based on the enrichment scores of immune-related gene signatures to identify immune subtypes. We described their immune characteristics and prognoses and conducted differential gene analysis and lasso regression to identify a prognostic biomarker, GZMM. For experimental validation, we performed immunohistochemical staining of GZMM in 39 p53-positive EC surgical samples. We defined two immune subtypes, immune-hot (IH) and immune-cold (IC), which differed in immune cell infiltration, cytokine and chemokine expression and prognosis. The IH subtype has significantly stronger immune activation than the IC subtype, showing a significant infiltration of immune effector cells and high expression of relevant chemokines, with better prognosis. Moreover, the immunohistochemical staining of GZMM in a cohort of 39 p53-positive EC surgical samples confirmed GZMM as a unique prognostic biomarker, with high expression in both tumor cells and lymphocytes predicting a better prognosis. Our study revealed heterogeneous immune subtypes in CNH EC and identified GZMM as a prognostic biomarker. The stratified classification strategy combining molecular and immune subtypes provides valuable insights for future clinical practice.

Deciphering the divergent transcriptomic landscapes of cervical cancer cells grown in 3D and 2D cell culture systems.

Cervical cancer remains a significant health challenge for women worldwide, with a disproportionate impact on developing regions like sub-Saharan Africa. Taking advantage of recent advancements in developing suitable preclinical models to study cell proliferation, differentiation, and gene expression, we used RNA sequencing to compare the transcriptomic profiles of SiHa cervical cancer cells grown in 3D versus 2D culture systems. Pathway analysis of 3D cultures revealed upregulation of immune activation, angiogenesis, and tissue remodeling pathways. The high expression of cytokines, chemokines, matrix metalloproteinases, and immediate early genes, suggests that 3D cultures replicate the tumor microenvironment better than 2D monolayer cultures. HPV gene expression analysis further demonstrated higher expression levels of HPV16 E1, E2, E6, and E7 genes in 3D versus 2D cultures. Further, by using a set of linear models, we identified 79 significantly differentially expressed genes in 3D culture compared to 2D culture conditions, independent of HPV16 viral gene effects. We subsequently validated five of these genes at the protein level in both the SiHa cell line and a newly developed, patient-derived cervical cancer cell line. In addition, correlation analysis identified 26 human genes positively correlated with viral genes across 2D and 3D culture conditions. The top five 3D versus 2D differentially expressed and HPV-correlated genes were validated via qRT-PCR in our patient derived cell line. Altogether, these findings suggest that 3D cultures provide superior model systems to explore mechanisms of immune evasion, cancer progression and antiviral therapeutics.

Analysis of the potential biological significance of glycosylation in triple-negative breast cancer on patient prognosis.

Breast cancer is the most common malignancy in women, with its prognosis varying greatly according to its subtype. Triple-negative breast cancer (TNBC) has the worst prognosis among all subtypes. Glycosylation is a critical factor influencing the prognosis of patients with TNBC. Our aim is to develop a tumor prognosis model by analyzing genes related to glycosylation to predict patient outcomes. The dataset used in this study was downloaded from the Cancer Genome Atlas Program (TCGA) database, and predictive genes were identified through Cox one-way regression analysis. The model genes with the highest risk scores among the 18 samples were obtained by lasso regression analysis to establish the model. We analyzed the pathways affecting the progression of TNBC and discovered key genes for subsequent research. Our model was constructed using data from TCGA database and validated through Kaplan-Meier curve analysis and Receiver Operating Characteristic (ROC) curve assessment. Our analysis revealed that a high expression of tumor-related chemokines in the high-risk group may be associated with poor tumor prognosis. Furthermore, we conducted a random survival forest analysis and identified two significant genes, namely DPM2 and PINK1, which have been selected for further investigation. The prognostic analysis model, developed based on the glycosylation genes in TNBC, exhibits excellent validation efficacy. This model is valuable for the prognostic analysis of patients with TNBC.

The Secretion of Inflammatory Cytokines Triggered by TLR2 Through Calcium‐Dependent and Calcium‐Independent Pathways in Keratinocytes

Keratinocytes can be activated by Cutibacterium acnes, leading to the production of proinflammatory cytokines via toll‐like receptors (TLRs) 2 and 4. Although several studies have investigated keratinocytes, the mechanism of calcium‐mediated activation remains unclear. Herein, we investigated whether calcium influx via TLR2 and TLR4 stimulation was involved in cytokine secretion by keratinocytes in HaCaT cells. Although TLR2 stimulation by peptidoglycan (PGN) increased intracellular calcium influx, TLR4 stimulation by lipopolysaccharide (LPS) did not increase it, as analyzed using flow cytometry with the calcium indicator Fluo‐3. However, activation by either TLR2 or TLR4 ligands upregulated the intracellular calcium influx in THP‐1 monocytes. Additionally, the expression of major proinflammatory cytokines and chemokines, such as interleukin (IL)‐6, IL‐8, IL‐1α, granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), and monocyte chemoattractant protein‐1 (MCP‐1), was significantly increased by TLR2 in HaCaT cells. Moreover, treatment with the intracellular calcium chelator, BAPTA‐AM, disrupted PGN‐mediated induction of IL‐6, IL‐8, and MCP‐1 production. Real‐time quantitative polymerase chain reaction (PCR) and western blotting revealed that TLR2 stimulation induced expression of the epidermal differentiation marker keratin 1. In conclusion, TLR2‐induced intracellular calcium influx plays a pivotal role in the secretion of proinflammatory cytokines, such as IL‐6 and MCP‐1, in keratinocytes. Moreover, the continuous influx of calcium via TLR2 activation leads to keratinization. In vitro studies using HaCaT cells provide basic research on the effect of TLR2‐induced calcium on C. acnes‐mediated inflammation in keratinocytes. These studies are limited in their ability to clinically predict what happens in human keratinocytes. Clinical studies on patients with acne, including three‐dimensional (3D) cultures of primary keratinocytes, are required to develop new diagnostic markers for determining the severity of acne vulgaris.

Breast cancer-derived CAV1 promotes lung metastasis by regulating integrin α6β4 and the recruitment and polarization of tumor-associated neutrophils.

Lung metastasis in breast cancer (BC) patients is one of the main reasons for their high mortality rate. The most prevalent BC small extracellular vesicles (sEVs receptor, integrin α6β4, has been found to interact with surfactant-associated protein (SFTPC) in lung epithelial cells, making BC more likely to metastasize to the lung. Tumor-associated neutrophils (TANs) play an essential role in BC lung metastasis as a component of the lung pre-metastatic niche (PMN) with two sides. It has been demonstrated that Toll-like Receptor4 (TLR4) can participate in signaling, such as NF-B and NLRP3, to facilitate tumor metastasis. A cellular membrane structural protein called caveolin-1 (CAV1) is associated with BC's proliferation, metastasis, and immunological control. According to our previous research, CAV1 on BC-derived sEVs facilitates the formation of the lung PMN by enhancing tenascin-C (TnC) secretion in lung fibroblasts to promote the deposition of ECM, by increasing the expression of PMN marker genes and inflammatory chemokines in lung epithelial cells, and by supporting N2-type polarization of lung macrophages via inhibiting the PTEN/CCL2/VEGF-A axis. More research is needed to determine how sEVs-mediated CAV1 facilitates BC-targeted metastasis to the lungs. By creating a stable-translocating cell line that stably interfered with CAV1 and a mouse model of BC lung metastasis, we investigated how sEVs-mediated CAV1 promotes BC lung metastasis and TAN recruitment and polarization in vivo and in vitro. In this study, we showed that CAV1 increases the likelihood that BC lung metastasis would occur by controlling the expression of integrin α6β4 and via boosting TANs recruitment and polarization through activating the TLR4-NF-B-IL-6/CCL2 and TLR4/NF-B/NLRP3 signaling pathways. According to our findings, CAV1 regulates integrin α6β4 and modulates TLR4 signaling, both of which are critical for BC lung metastasis. This finding may open new avenues for BC lung metastasis prevention and treatment.