The ADAM17 inhibitor ZLDI-8 sensitized hepatocellular carcinoma cells to sorafenib through Notch1-integrin ß-talk.

ZLDI-8 is an A disintegrin and metalloproteinase domain 17 (ADAM17) inhibitor that suppresses the shedding of Notch1 to the Notch1 intracellular domain (NICD). In previous studies, we found that ZLDI-8 was able to sensitize HCC to sorafenib, but the mechanism of action remains unclear. The sensitizing effects of ZLDI-8 were tested both in vitro and in vivo. EMT-related factors, sorafenib sensitivity-related proteins and ECM-related gene expression were assessed using immunohistochemistry, RTPCR and Western blotting. Knockdown assays were conducted to determine the relationship between the Notch and Integrin pathways. CoIP assays, nuclear and cytoplasmic fractionation and immunofluorescence colocalization were applied to explore the interaction between the Notch and Integrin pathways. Appropriate statistical analysis methods were used to assess the significance of the experimental results and to ensure the scientific validity and reliability of the experimental design. We found that ECM- and EMT-related proteins were downregulated after ZLDI-8 treatment (P<0.05). ZLDI-8 significantly downregulated Integrinß1 and Integrinß3 in HCC in vitro and in vivo (P<0.05), possibly through Foxc2-dependent regulation. Mechanistically, interfering with the expression of both Integrin-linked kinase (ILK) and the NICD may downregulate the expression of proteins targeted by sorafenib, thereby sensitizing cells to sorafenib. The retroregulation of Integrinß by ILK may occur through the interaction between the NICD and ILK and may be the result of the translocation of the complexus. Our study indicates that blocking the Notch pathway may affect Integrinß through crosstalk between the Notch1 and Integrinß/ILK signaling pathways, thus providing a potential therapeutic strategy for HCC.

Exploring the role of ITGB6: fibrosis, cancer, and other diseases.

Integrin ß6 (ITGB6), a member of the integrin family of proteins, is only present in epithelial tissues and frequently associates with integrin subunit αv to form transmembrane heterodimers named integrin αvß6. Importantly, ITGB6 determines αvß6 expression and availability. In addition to being engaged in organ fibrosis, ITGB6 is also directly linked to the emergence of cancer, periodontitis, and several potential genetic diseases. Therefore, it is of great significance to study the molecular-biological mechanism of ITGB6, which could provide novel insights for future clinical diagnosis and therapy. This review introduces the structure, distribution, and biological function of ITGB6. This review also expounds on ITGB6-related diseases, detailing the known biological effects of ITGB6.

CircItgb5 promotes synthetic phenotype of pulmonary artery smooth muscle cells via interacting with miR-96-5p and Uba1 in monocrotaline-induced pulmonary arterial hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a rare but fatal cardiopulmonary disease mainly characterized by pulmonary vascular remodeling. Aberrant expression of circRNAs has been reported to play a crucial role in pulmonary vascular remodeling. The existing literature predominantly centers on studies that examined the sponge mechanism of circRNAs. However, the mechanism of circRNAs in regulating PAH-related protein remains largely unknown. This study aimed to investigate the effect of circItgb5 on pulmonary vascular remodeling and the underlying functional mechanism. MATERIALS AND METHODS: High-throughput circRNAs sequencing was used to detect circItgb5 expression in control and PDGF-BB-treated pulmonary arterial smooth muscle cells (PASMCs). Localization of circItgb5 in PASMCs was determined via the fluorescence in situ hybridization assay. Sanger sequencing was applied to analyze the circularization of Itgb5. The identification of proteins interacting with circItgb5 was achieved through a RNA pull-down assay. To assess the impact of circItgb5 on PASMCs proliferation, an EdU assay was employed. Additionally, the cell cycle of PASMCs was examined using a flow cytometry assay. Western blotting was used to detect biomarkers associated with the phenotypic switch of PASMCs. Furthermore, a monocrotaline (MCT)-induced PAH rat model was established to explore the effect of silencing circItgb5 on pulmonary vascular remodeling. RESULTS: CircItgb5 was significantly upregulated in PDGF-BB-treated PASMCs and was predominately localized in the cytoplasm of PASMCs. In vivo experiments revealed that the knockdown of circItgb5 attenuated MCT-induced pulmonary vascular remodeling and right ventricular hypertrophy. In vitro experiments revealed that circItgb5 promoted the transition of PASMCs to synthetic phenotype. Mechanistically, circItgb5 sponged miR-96-5p to increase mTOR level and interacted with Uba1 protein to activate the Ube2n/Mdm2/ACE2 pathway. CONCLUSIONS: CircItgb5 promoted the transition of PASMCs to synthetic phenotype by interacting with miR-96-5p and Uba1 protein. Knockdown of circItgb5 mitigated pulmonary arterial pressure, pulmonary vascular remodeling and right ventricular hypertrophy. Overall, circItgb5 has the potential for application as a therapeutic target for PAH.

Upregulation of ITGB6 in primary palmar hyperhidrosis.

BACKGROUND: The regulatory effect of integrin ß6 (ITGB6) on sweat gland cells in primary palmar hyperhidrosis (PPH) remains unclear. OBJECTIVES: This study investigated the involvement of ITGB6 in the pathogenesis of PPH. MATERIAL AND METHODS: Sweat gland tissues were collected from PPH patients and healthy volunteers. The expression levels of ITGB6 in sweat gland tissues were detected with quantitative polymerase chain reaction (qPCR), western blot and immunohistochemical staining. Sweat gland cells were extracted from PPH patients, and identified with immunofluorescence staining of CEA and CK7. The expression of aquaporin 5 (AQP5) and Na-K-Cl cotransporter 1 (NKCC1) in primary sweat gland cells that overexpress ITGB6 were also detected. Through a series of bioinformatic methods, differentially expressed genes in sweat gland tissues were examined and validated via comparing PPH samples and controls. The key proteins and biological functions enriched in PPH were determined using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. RESULTS: The ITGB6 was upregulated in sweat gland tissues of PPH patients compared to that of healthy volunteers. The CEA and CK7 were positively expressed in sweat gland cells extracted from PPH patients. The overexpression of ITGB6 upregulated AQP5 and NKCC1 protein expression in the sweat gland cells of PPH patients. A total of 562 differentially expressed mRNAs were identified using high-throughput sequencing (394 upregulated, 168 downregulated), which were mainly active in the chemokine and Wnt signaling pathways. After verification with qPCR and western blot, the overexpression of ITGB6 significantly upregulated CXCL3, CXCL5, CXCL10, and CXCL11, and downregulated Wnt2 mRNA and protein expression in sweat gland cells. CONCLUSIONS: The ITGB6 is upregulated in PPH patients. It may be involved in the pathogenesis of PPH by upregulating AQP5, NKCC1, CXCL3, CXCL5, CXCL10, and CXCL11, and downregulating Wnt2 expression in sweat glands.

Integrin subunit beta 8 contributes to lenvatinib resistance in HCC.

Lenvatinib is a multikinase inhibitor approved as a first-line therapy for advanced hepatocellular carcinoma (HCC). However, the development of drug resistance is common, and the underlying mechanisms governing this resistance are largely unknown. In this study, we established two lenvatinib-resistant (LR) HCC cell lines and identified integrin subunit beta 8 (ITGB8) as a critical contributor to lenvatinib resistance in HCC. The elevated expression of ITGB8 was observed in LR HCC cells. Furthermore, silencing of ITGB8 reversed lenvatinib resistance in vitro and in vivo, whereas ectopic expression of ITGB8 in lenvatinib-sensitive parental HCC cells exhibited increased resistance to lenvatinib. Mechanistically, ITGB8 regulated lenvatinib resistance through an HSP90-mediated stabilization of AKT and enhanced AKT signaling. In support of this model, either an AKT inhibitor MK-2206 or an HSP90 inhibitor 17-AAG resensitized LR HCC cells to lenvatinib treatment. Conclusion: Collectively, our results establish a crucial role of ITGB8 in lenvatinib resistance, and suggest that targeting the ITGB8/HSP90/AKT axis is a promising therapeutic strategy in patients with HCC exhibiting lenvatinib resistance.

The Alleviating Effect of Lagerstroemia indica Flower Extract on Stretch Marks through Regulation of Mast Cells.

Striae distensae (SD) or stretch marks are common linear scars of atrophic skin with disintegrating extracellular matrix (ECM) structures. Although fibroblasts contribute to the construction of ECM structure in SD, some studies have reported that mast cell degranulation causes the disruption of ECM in early SD lesions. Lagerstroemia indica flower (LIF) has traditionally been used in India as a diuretic. However, little is known about the effect and molecular action of Lagerstroemia indica flower extract (LIFE) on alleviating SD. This study evaluated the effects of LIFE on mast cell degranulation and the synthesis of ECM components in fibroblasts. LIFE inhibits the adhesion of rat basophilic leukemia (RBL) cells, RBL-2H3 on fibronectin (FN) and the expression of integrin, a receptor for FN, thereby reducing focal adhesion kinase (FAK) phosphorylation. In addition, LIFE attenuated the allergen-induced granules and cytokine interleukin 3 (IL-3) through the adhesion with FN. Moreover, the conditioned medium (CM) of activated mast cells decreases the synthesis of ECM components, and LIFE restores the abnormal expressions induced by activated mast cells. These results demonstrate that LIFE suppresses FN-induced mast cell activation and promotes the synthesis of ECM components in fibroblast, which indicates that LIFE may be a useful cosmetic agent for SD treatment.

The ß8 integrin cytoplasmic domain activates extracellular matrix adhesion to promote brain neurovascular development.

In the developing mammalian brain, neuroepithelial cells interact with blood vessels to regulate angiogenesis, blood-brain barrier maturation and other key neurovascular functions. Genetic studies in mice have shown that neurovascular development is controlled, in part, by Itgb8, which encodes the neuroepithelial cell-expressed integrin ß8 subunit. However, these studies have involved complete loss-of-function Itgb8 mutations, and have not discerned the relative roles for the ß8 integrin extracellular matrix (ECM) binding region versus the intracellular signaling tail. Here, Cre/lox strategies have been employed to selectively delete the cytoplasmic tail of murine Itgb8 without perturbing its transmembrane and extracellular domains. We report that the ß8 integrin cytoplasmic domain is essential for inside-out modulation of adhesion, including activation of latent-TGFßs in the ECM. Quantitative sequencing of the brain endothelial cell transcriptome identifies TGFß-regulated genes with putative links to blood vessel morphogenesis, including several genes linked to Wnt/ß-catenin signaling. These results reveal that the ß8 integrin cytoplasmic domain is essential for the regulation of TGFß-dependent gene expression in endothelial cells and suggest that cross-talk between TGFßs and Wnt pathways is crucial for neurovascular development.

Integrin αvß6 contributes to the development of intestinal fibrosis via the FAK/AKT signaling pathway.

Intestinal fibrosis is one of the most severe complications of inflammatory bowel disease (IBD) and frequently requires surgery due to intestinal obstruction. Integrin αvß6, which is mainly regulated by the integrin ß6 subunit gene (ITGB6), is a special integrin subtype expressed only in epithelial cells. In our previous study, we found integrin αvß6 can promote the development of IBD, but the role of integrin αvß6 in intestinal fibrosis remains unclear. In this study, we observed a gradual increase of ITGB6 mRNA expression from normal region to stenotic region of IBD patients' intestinal specimens. Next, we established a dextran sulfate sodium (DSS)-induced intestinal fibrosis model and a heterotopic intestinal transplant model, and found intestinal fibrosis was decreased in ITGB6-deficient mice compared to wild-type (WT) mice. Furthermore, we performed RNA-sequencing and KEGG pathway analysis on intestinal tissues from ITGB6-overexpressing transgenic mice and WT mice, and found multiple pathways containing ITGB6, are related to the activation of focal adhesion kinase (FAK); finding was confirmed by Western blot. At last, we generated a heterotopic intestinal transplant model found the FAK/AKT pathway was inhibited in ITGB6-deficient mice. In conclusion, our data demonstrate that integrin αvß6 promotes the pathogenesis of intestinal fibrosis by FAK/AKT pathway, making integrin αvß6 a potential therapeutic target to prevent this condition.

Peri-implantation expression and regulation of ITGB8 in goat uterus.

Ruminants have a superficial implantation pattern. The extended conceptus attaches to the receptive endometrium to form the cotyledonary placenta. During the attachment, a large number of events occur at the maternal-fetal interface. However, the related molecular mechanisms have not been fully understood. Integrin beta8 (ITGB8) is a subunit of integrin beta involved in embryo implantation. In this study, we determined peri-implantation expression and regulation of ITGB8 in goat uterus. The mRNA and protein levels of ITGB8 were both high in goat endometrial luminal epithelium (LE) and superficial glandular epithelium (sGE) during the adhesion period (Days 16-19 of pregnancy). Such expression profile was opposite to that of microRNA-187 (miR-187). Then, we validated that miR-187 targeted the 3' untranslated region (UTR) of ITGB8 in primary goat endometrial epithelial cells (EECs). In EECs, inhibition of miR-187 resulted in not only up-regulated ITGB8 level but also reduced cell proliferation and focal adhesion kinase (FAK) activity. Moreover, ITGB8 and miR-187 were regulated by interferon tau (IFNT). Altogether, in goat, the miR-187/ITGB8 axis may be involved in conceptus attachment and is downstream of IFNT. Our results will help us better understand the mechanisms of ruminant implantation and may provide a useful tool to improve the reproduction ratio for ruminants.

ITGB6 inhibits the proliferation of porcine skeletal muscle satellite cells.

The formation of embryonic muscle fibers determines the amount of postnatal muscles and is regulated by a variety of signaling pathways and transcription factors. Previously, by using chromatin immunoprecipitation-sequencing and RNA-Seq techniques, we identified a large number of genes that are regulated by H3K27me3 in porcine embryonic skeletal muscles. Among these genes, we found that ITGB6 is regulated by H3K27me3. However, its function in muscle development is unknown. In this study, we first verified that ITGB6 was differentially regulated by H3K27me3 and that its expression levels were upregulated in porcine skeletal muscles at embryonic Days 33, 65, and 90. Then, we performed gain- or loss-of-function studies on porcine skeletal muscle satellite cells to study the role of ITGB6 in porcine skeletal muscle development. The proliferation of porcine skeletal muscle satellite cells was studied through real-time polymerase chain reaction, Cell Counting Kit-8, 5-ethynyl-2'-deoxyuridine staining, Western blot, and flow cytometry analyses. We found that the ITGB6 gene was regulated by H3K27me3 during muscle development and had an inhibitory effect on the proliferation of porcine skeletal muscle satellite cells.

Antibody secreting cells are critically dependent on integrin α4ß7/MAdCAM-1 for intestinal recruitment and control of the microbiota during chronic colitis.

T and B cells employ integrin α4ß7 to migrate to intestine under homeostatic conditions. Whether those cells differentially rely on α4ß7 for homing during inflammatory conditions has not been fully examined. This may have implications for our understanding of the mode of action of anti-integrin therapies in inflammatory bowel disease (IBD). Here, we examined the role of α4ß7 integrin during chronic colitis using IL-10-/- mice, ß7-deficient IL-10-/-, IgA-deficient IL-10-/- mice, and antibody blockade of MAdCAM-1. We found that α4ß7 was predominantly expressed by B cells. ß7 deficiency and MAdCAM-1 blockade specifically depleted antibody secreting cells (ASC) (not T cells) from the colonic LP, leading to a fecal pan-immunoglobulin deficit, severe colitis, and alterations of microbiota composition. Colitis was not due to defective regulation, as dendritic cells (DC), regulatory T cells, retinaldehyde dehydrogenase (RALDH) expression, activity, and regulatory T/B-cell cytokines were all comparable between the strains/treatment. Finally, an IgA deficit closely recapitulated the clinical phenotype and altered microbiota composition of ß7-deficient IL-10-/- mice. Thus, a luminal IgA deficit contributes to accelerated colitis in the ß7-deficient state. Given the critical/nonredundant dependence of IgA ASC on α4ß7:MAdCAM-1 for intestinal homing, B cells may represent unappreciated targets of anti-integrin therapies.

The RGD (Arg-Gly-Asp) is a potential cell-binding motif of UNC-52/PERLECAN.

UNC-52/perlecan is a basement membrane (BM) proteoglycan playing an essential role in the muscle cell attachment of C. elegans. The UNC-52 protein contains two RGD (Arg-Gly-Asp) motifs in domains III and IV, a well-characterized tripeptide known for binding to mammalian ß integrin. To investigate the role of the RGD motif in UNC-52/perlecan, we created two mutations in the 2021RGD2023 motif: one mutation changed the RGD to an RGE, and the other deleted the RGD motif. The RGE2023 caused defective actin filaments and aberrant localization of PAT-3 ß integrin and TLN-1/talin. Additionally, the in-frame deletion of RGD2023 resulted in a paralyzed and arrested at two-fold embryonic stages (Pat) phenotype, which is the identical phenotype of the pat-3 ß integrin null allele. These results indicate that RGD2023 is a potential ligand for cell binding and is essential for development and survival. Furthermore, our analysis reveals that the RGD of an invertebrate BM molecule is a potential cell-binding motif, suggesting that the function of the RGD motif is conserved among species.

Scavenger receptor MARCO contributes to macrophage phagocytosis and clearance of tumor cells.

Macrophage receptor with collagenous structure (MARCO) is a member of the class A scavenger receptor family which is expressed on the cell surface of macrophages. It is well known that MARCO avidly binds to unopsonized pathogens, leading to its ingestion by macrophages. However, the role of MARCO in the recognition and phagocytosis of tumor cells by macrophages remains poorly understood. In this study, we used lentiviral technology to knockdown and overexpress MARCO and investigated the ability of macrophages to phagocytose tumor cells. Our results showed that MARCO expression was correlated with the ability of macrophages to carry on phagocytosis. MARCO knockdown led to significant decreases in the number of engulfment pseudopodia of macrophages and inhibition of the phagocytosis of tumor cells. On the other hand, MARCO overexpression elevated activity of SYK, PI3K and Rac1 in macrophages, which led to changes in macrophage morphology and enhanced phagocytosis by promoting formation of stress fibers and pseudopodia. By Co-IP analysis we showed that MARCO directly binds to the ß5 integrin of SL4 tumor cells. In summary, these results demonstrated the important role for MARCO in demonstrated tumor cells uptake and clearance by macrophages.

Dynamics and epigenetic signature of regulatory T-cells following antiretroviral therapy initiation in acute HIV infection.

BACKGROUND: HIV infection promotes the expansion of immunosuppressive regulatory T-cells (Tregs), contributing to immune dysfunction, tissue fibrosis and disease progression. Early antiretroviral treatment (ART) upon HIV infection improves CD4 count and decreases immune activation. However, Treg dynamics and their epigenetic regulation following early ART initiation remain understudied. METHODS: Treg subsets were characterized by flow cytometry in 103 individuals, including untreated HIV-infected participants in acute and chronic phases, ART-treated in early infection, elite controllers (ECs), immunological controllers (ICs), and HIV-uninfected controls. The methylation status of six regulatory regions of the foxp3 gene was assessed using MiSeq technology. FINDINGS: Total Treg frequency increased overtime during HIV infection, which was normalized in early ART recipients. Tregs in untreated individuals expressed higher levels of activation and immunosuppressive markers (CD39, and LAP(TGF-ß1)), which remained unchanged following early ART. Expression of gut migration markers (CCR9, Integrin-ß7) by Tregs was elevated during untreated HIV infection, while they declined with the duration of ART but not upon early ART initiation. Notably, gut-homing Tregs expressing LAP(TGF-ß1) and CD39 remained higher despite early treatment. Additionally, the increase in LAP(TGF-ß1)+ Tregs overtime were consistent with higher demethylation of conserved non-coding sequence (CNS)-1 in the foxp3 gene. Remarkably, LAP(TGF-ß1)-expressing Tregs in ECs were significantly higher than in uninfected subjects, while the markers of Treg activation and gut migration were not different. INTERPRETATION: Early ART initiation was unable to control the levels of immunosuppressive Treg subsets and their gut migration potential, which could ultimately contribute to gut tissue fibrosis and HIV disease progression. FUNDING: This study was funded by the Canadian Institutes of Health Research (CIHR, grant MOP 142294) and in part by the AIDS and Infectious Diseases Network of the Réseau SIDA et maladies infectieuses du Fonds de recherche du Québec-Santé (FRQ-S).

Temperature response of enriched pre-pubertal caprine male germline stem cells in vitro.

The present study aims to evaluate culture temperature-dependent variation in survival, growth characteristics and expression of stress, pluripotency, apoptosis, and adhesion markers in enriched caprine male germline stem cells (cmGSCs). For this, testes from pre-pubertal bucks (4-5 months; n = 4) were used to isolated cells by a two-step enzymatic digestion method. After enrichment of cmGSCs by multiple methods (differential platting, Percoll density gradient centrifugation, and MACS), viability of CD90+ cells was assessed before co-cultured onto the Sertoli cell feeder layer at different temperatures (35.5, 37.0, 38.5, and 40.0 °C). The culture characteristics of cells were compared with MTT assay (viability); cluster-forming activity assay, SA-ß1-gal assay (senescence), BrdU assay (proliferation), and transcript expression analyses by qRT-PCR. Moreover, the co-localization of pluripotency markers (UCHL-1, PLZF, and DBA) was examined by a double-immunofluorescence method. The cells grown at 37.0 °C showed faster proliferation with a significantly (p < 0.05) higher number of viable cells and greater number of cell clusters, besides higher expression of pluripotency markers. The transcript expression of HSPs (more noticeably HSP72 than HSP73), anti-oxidative enzymes (GPx and CuZnSOD), and adhesion molecule (ß1-integrin) was significantly (p < 0.05) downregulated when grown at 35.0, 38.5, or 40.0 °C compared with 37.0 °C. The expression of pluripotency-specific transcripts was significantly (p < 0.05) lower in cmGSCs grown at the culture temperature lower (35.5 °C) or higher (38.5 °C and 40.0 °C) than 37.0 °C. Overall, the culture temperature significantly affects the proliferation, growth characteristics, and expression of heat stress, pluripotency, and adhesion-specific markers in pre-pubertal cmGSCs. These results provide an insight to develop strategies for the improved cultivation and downstream applications of cmGSCs.

Regulation and Role of αE Integrin and Gut Homing Integrins in Migration and Retention of Intestinal Lymphocytes during Inflammatory Bowel Disease.

Targeting interactions between α4ß7 integrin and endothelial adhesion molecule MAdCAM-1 to inhibit lymphocyte migration to the gastrointestinal tract is an effective therapy in inflammatory bowel disease (IBD). Following lymphocyte entry into the mucosa, a subset of these cells expresses αEß7 integrin, which is expressed on proinflammatory lymphocytes, to increase cell retention. The factors governing lymphocyte migration into the intestinal mucosa and αE integrin expression in healthy subjects and IBD patients remain incompletely understood. We evaluated changes in factors involved in lymphocyte migration and differentiation within tissues. Both ileal and colonic tissue from active IBD patients showed upregulation of ICAM-1, VCAM-1, and MAdCAM-1 at the gene and protein levels compared with healthy subjects and/or inactive IBD patients. ß1 and ß7 integrin expression on circulating lymphocytes was similar across groups. TGF-ß1 treatment induced expression of αE on both ß7+ and ß7- T cells, suggesting that cells entering the mucosa independently of MAdCAM-1/α4ß7 can become αEß7+ ITGAE gene polymorphisms did not alter protein induction following TGF-ß1 stimulation. Increased phospho-SMAD3, which is directly downstream of TGF-ß, and increased TGF-ß-responsive gene expression were observed in the colonic mucosa of IBD patients. Finally, in vitro stimulation experiments showed that baseline ß7 expression had little effect on cytokine, chemokine, transcription factor, and effector molecule gene expression in αE+ and αE- T cells. These findings suggest cell migration to the gut mucosa may be altered in IBD and α4ß7-, and α4ß7+ T cells may upregulate αEß7 in response to TGF-ß once within the gut mucosa.

ITGB6-Knockout Suppresses Cholangiocarcinoma Cell Migration and Invasion with Declining PODXL2 Expression.

Intrahepatic cholangiocarcinoma (iCCA) is a heterogeneous bile duct cancer with a poor prognosis. Integrin αvß6 (ß6) has been shown to be upregulated in iCCA and is associated with its subclassification and clinicopathological features. In the present study, two ITGB6-knockout HuCCT1 CCA cell lines (ITGB6-ko cells) were established using the clustered regulatory interspaced short palindromic repeats (CRISPR), an associated nuclease 9 (Cas9) system, and single-cell cloning. RNA sequencing analysis, real-time polymerase chain reaction (PCR), and immunofluorescent methods were applied to explore possible downstream factors. ITGB6-ko cells showed significantly decreased expression of integrin ß6 on flow cytometric analysis. Both cell lines exhibited significant inhibition of cell migration and invasion, decreased wound-healing capability, decreased colony formation ability, and cell cycle dysregulation. RNA sequencing and real-time PCR analysis revealed a remarkable decrease in podocalyxin-like protein 2 (PODXL2) expression in ITGB6-ko cells. Colocalization of PODXL2 and integrin ß6 was also observed. S100 calcium-binding protein P and mucin 1, which are associated with CCA subclassification, were downregulated in ITGB6-ko cells. These results describe the successful generation of ITGB6-ko CCA cell clones with decreased migration and invasion and downregulation of PODXL2, suggesting the utility of integrin ß6 as a possible therapeutic target or diagnostic marker candidate.

Conformation-sensitive targeting of lipid nanoparticles for RNA therapeutics.

The successful in vivo implementation of gene expression modulation strategies relies on effective, non-immunogenic delivery vehicles. Lipid nanoparticles are one of the most advanced non-viral clinically approved nucleic-acid delivery systems. Yet lipid nanoparticles accumulate naturally in liver cells upon intravenous administration, and hence, there is an urgent need to enhance uptake by other cell types. Here we use a conformation-sensitive targeting strategy to achieve in vivo gene silencing in a selective subset of leukocytes and show potential therapeutic applications in a murine model of colitis. In particular, by targeting the high-affinity conformation of α4ß7 integrin, which is a hallmark of inflammatory gut-homing leukocytes, we silenced interferon-γ in the gut, resulting in an improved therapeutic outcome in experimental colitis. The lipid nanoparticles did not induce adverse immune activation or liver toxicity. These results suggest that our lipid nanoparticle targeting strategy might be applied for selective delivery of payloads to other conformation-sensitive targets.

Integrated analysis reveals the participation of IL4I1, ITGB7, and FUT7 in reshaping the TNBC immune microenvironment by targeting glycolysis.

BACKGROUND: The overall response rate of immunotherapy in triple-negative breast cancer (TNBC) remains unsatisfactory. Accumulating evidence indicated that glucose metabolic reprogramming could modulate immunotherapy efficacy. However, transcriptomic evidence remains insufficient. METHODS: Genes' relationship with glucose metabolism and TNBC-specific immune was demonstrated by weighted gene co-expression network analysis (WGCNA). The glucose metabolic capability was estimated by standardised uptake value (SUV), an indicator of glucose uptake in 18 F-fluorodeoxyglucose positron emission tomography (FDG-PET), and a reflection of cancer metabolic behaviour. PD-(L)1 expression was used to reflect the efficacy of immunotherapy. Additionally, immune infiltration, survival, and gene coexpression profiles were provided. RESULTS: Comprehensive analysis revealing that IL4I1, ITGB7, and FUT7 hold the potential to reinforce immunotherapy by reshaping glucose metabolism in TNBC. These results were verified by functional enrichment analysis, which demonstrated their relationships with immune-related signalling pathways and extracellular microenvironment reprogramming. Their expressions have potent positive correlations with Treg and Macrophage cell infiltration and exhausted T cell markers. Meanwhile, their overexpression also lead to poor prognosis. CONCLUSION: IL4I1, ITGB7, and FUT7 may be the hub genes that link glucose metabolism, and cancer-specific immunity. They may be potential targets for enhancing ICB treatment by reprogramming the tumour microenvironment and remodelling tumour metabolism.

Nonpeptidergic neurons suppress mast cells via glutamate to maintain skin homeostasis.

Cutaneous mast cells mediate numerous skin inflammatory processes and have anatomical and functional associations with sensory afferent neurons. We reveal that epidermal nerve endings from a subset of sensory nonpeptidergic neurons expressing MrgprD are reduced by the absence of Langerhans cells. Loss of epidermal innervation or ablation of MrgprD-expressing neurons increased expression of a mast cell gene module, including the activating receptor, Mrgprb2, resulting in increased mast cell degranulation and cutaneous inflammation in multiple disease models. Agonism of MrgprD-expressing neurons reduced expression of module genes and suppressed mast cell responses. MrgprD-expressing neurons released glutamate which was increased by MrgprD agonism. Inhibiting glutamate release or glutamate receptor binding yielded hyperresponsive mast cells with a genomic state similar to that in mice lacking MrgprD-expressing neurons. These data demonstrate that MrgprD-expressing neurons suppress mast cell hyperresponsiveness and skin inflammation via glutamate release, thereby revealing an unexpected neuroimmune mechanism maintaining cutaneous immune homeostasis.