Interaction of the Immune System TIM-3 Protein with a Model Cellular Membrane Containing Phosphatidyl-Serine Lipids.

T cell transmembrane, Immunoglobulin, and Mucin (TIM) are important immune system proteins which are especially present in T-cells and regulated the immune system by sensing cell engulfment and apoptotic processes. Their role is exerted by the capacity to detect the presence of phosphatidyl-serine lipid polar head in the outer leaflet of cellular membranes (correlated with apoptosis). In this contribution by using equilibrium and enhanced sampling molecular dynamics simulation we unravel the molecular bases and the thermodynamics of TIM, and in particular TIM-3, interaction with phosphatidyl serine in a lipid bilayer. Since TIM-3 deregulation is an important factor of pro-oncogenic tumor micro-environment understanding its functioning at a molecular level may pave the way to the development of original immunotherapeutic approaches.

Soluble T cell immunoglobulin and mucin-domain containing protein 3 in children hospitalized with pneumonia in resource-limited settings.

In a prospective cohort study of 77 children with severe pneumonia from two hospitals in Uganda, we assessed soluble T cell immunoglobulin and mucin-domain containing protein 3 (sTIM-3) levels at hospital admission and their association with pneumonia severity and subsequent mortality. sTIM-3 levels were positively correlated with the Respiratory Index of Severity in Children (RISC) (ρ = 0.35, p = 0.0017), sTIM-3 levels were higher in children who required transfer to a tertiary hospital (p = 0.014) and in fatal cases (p = 0.011). In summary, sTIM-3 is associated with disease severity and predictive of mortality in childhood pneumonia in resource-limited settings.

MUC3A promotes the progression of colorectal cancer through the PI3K/Akt/mTOR pathway.

Mucin 3A (MUC3A) is overexpressed in colorectal cancer (CRC) and associated with poor prognosis, but the related mechanism remains unclear. Our study found that MUC3A promotes the progression of CRC by activating the PI3K/Akt/mTOR signaling pathway. Knockout of MUC3A significantly inhibited the proliferation of CRC cells and induced G1 phase arrest by upregulating p21 protein, an important cell cycle regulator. Moreover, knockout of MUC3A significantly inhibited invasion ability and enhanced the sensitivity to the chemotherapeutic agent 5-FU. Furthermore, we found that knockout of MUC3A repressed the PI3K/Akt/mTOR pathway through RNA-seq. Treatment with the PI3K/Akt/mTOR pathway inhibitor rapamycin successfully eliminated the difference in proliferation, invasion and chemoresistance between MUC3A knockout cells and control cells. Our study suggests that MUC3A is a potential oncogene that promotes the proliferation, invasion, and chemotherapy resistance of CRC. Moreover, CRC patients with high expression of MUC3A may benefit from rapamycin treatment.

Serum T Cell Immunoglobulin Mucin 3 Predicts Worse Prognosis in Hepatocellular Carcinoma Patients Undergoing Transcatheter Arterial Chemoembolization.

BACKGROUND Hepatocellular carcinoma (HCC) is currently a leading cause of cancer-related death, and its prognostic evaluation remains a challenge. We aimed to explore the predictive value of T cell immunoglobulin and mucin 3 (Tim-3) in HCC patients undergoing transcatheter arterial chemoembolization (TACE). MATERIAL AND METHODS For the present study, 167 HCC patients who had undergone TACE were retrospectively recruited and randomly stratified into 2 subgroups at a ratio of about 2: 1 (training: 109; validation: 58) using EXCEL software. Pre-TACE serum was collected from all of these patients, and in a second visit a month after the initial treatment, an additional serum sample was collected from 45 patients in the training set. Tim-3 concentrations were determined by enzyme-linked immunosorbent assay. Kaplan-Meier survival curves analysis, log-rank tests, and Cox proportional hazard regression model were applied to evaluate prognostic significance. RESULTS Patients whose cancer progressed after TACE had significantly higher serum Tim-3 in both the training and validation sets (both P<0.05). Kaplan-Meier analysis revealed that patients with elevated serum Tim-3 had significantly shorter time-to-progression and overall survival in both the training and validation sets (both P<0.05). Multivariate Cox regression analysis confirmed that elevated pre-TACE Tim-3 served as a novel, independent indicator for predicting poor prognosis in HCC [progression, hazard ratio (HR) 2.197; 95% confidential interval (CI) 1.408-3.373; P<0.001; death, HR 2.570; 95% CI 1.508-4.378; P=0.001], which was further verified in the validation set (progression P=0.005; death P=0.043). Interestingly, serum Tim-3 retained its prognostic significance in the a-fetoprotein-negative subgroup. Notably, patients with high Tim-3 levels after TACE encountered dismal outcomes. CONCLUSIONS Serum Tim-3 might be a satisfactory prognostic indicator for HCC patients undergoing TACE.

The Detection of Immunity against WT1 and SMAD4P130L of EpCAM+ Cancer Cells in Malignant Pleural Effusion.

Malignant pleural effusion (MPE) provides a liquid tumor microenvironment model that includes cancer cells and immune cells. However, the characteristics of tumor antigen-specific CD8+ T cells have not been investigated in detail. Here, we analyzed MPE samples taken from a patient with pancreatic cancer who received a dendritic cell vaccine targeting Wilms' Tumor 1 (WT1) antigen over the disease course (two points at MPE1st and 2nd, two months after MPE1st). Epithelial cell adhesion molecule (EpCAM)+ cancer cells (PD-L1- or T cell immunoglobulin mucin-3, TIM-3-), both PD-1 or TIM-3 positive CD8+ T cells, and CD14+CD68+CD163+TIM-3+ macrophages increased from the MPE1st to MPE2nd. The ratio of WT1-specific cytotoxic lymphocytes (WT1-CTLs) to MPE CD8+ T cells and IFN-γ secretion of WT1-CTLs were reduced with disease progression. Coincidentally, the fraction of central memory T (TCM) of WT1-CTLs was decreased. On the other hand, CD8+ T cells in response to SMAD4P130L, which is homogeneously expressed in EpCAM+ cancer cells, were detected using in vitro expansion with the HLA-A*11:01 restrictive SVCVNLYH neoantigen. Furthermore, the CD8+ T cell response to SMAD4P130L was diminished following remarkably decreased numbers of CD8+ TCM in MPE samples. In conclusion, CD8+ T cells responding to WT1 or SMAD4P130L neoantigen expressed in EpCAM+ pancreatic cancer cells were detected in MPE. A tumor antigen-specific immune response would provide novel insight into the MPE microenvironment.

SdsA1, a secreted sulfatase, contributes to the in vivo virulence of Pseudomonas aeruginosa in mice.

Mucin is a glycoprotein that is the primary component of the mucus overlaying the epithelial tissues. Because mucin functions as a first line of the innate immune system, Pseudomonas aeruginosa appears to require interaction with mucin to establish infection in the host. However, the interactions between P. aeruginosa and mucin have been poorly understood. In this study, using in vivo expression technology (IVET), we attempted to identify mucin-inducible promoters that are likely to be involved in the establishment of P. aeruginosa infection. The IVET analysis revealed that the genes encoding glycosidases, sulfatases, and peptidases that are thought to be required for the utilization of mucin as a nutrient are present in 13 genes downstream of the identified promoters. Our results indicated that, among them, sdsA1 encoding a secreted sulfatase plays a central role in the degradation of mucin. It was then demonstrated that disruption of sdsA1 leads to a decreased release of sulfate from mucin and sulfated sugars. Furthermore, the sdsA1 mutant showed a reduction in the ability of mucin gel penetration and an attenuation of virulence in leukopenic mice compared with the wild-type strain. Collectively, these results suggest that SdsA1 plays an important role as a virulence factor of P. aeruginosa.

[The modulatory activity of T cell immunoglobulin and mucin domain-containing protein 3 on T lymphocytes in patients with chronic heart failure].

Objective: To investigate the expression of T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) in patients with chronic heart failure and its modulatory activity on T lymphocytes. Methods: Eighty-six patients with chronic heart failure (CHF group) who were hospitalized in Department of Cardiology, the First Affiliated Hospital of Zhengzhou University between January and October 2018 were enrolled in the study. Meanwhile, thirty-two healthy controls (HC group) who received healthy examination were also selected. Peripheral blood mononuclear cells were isolated, and TIM-3 expression of CD4(+)and CD8(+)T cells was investigated by flow cytometry. CD4(+)and CD8(+)T cells were purified, and were stimulated by anti-TIM-3 antibody. Interferon-γ (IFN-γ), interleukin (IL)-4, IL-10, IL-35, IL-17, and IL-22 expressions in the supernatants of cultured CD4(+)T cells and tumor necrosis factor-α (TNF-α) and IFN-γ expressions in the supernatants of cultured CD8(+) T cells were measured by enzyme-linked immunosorbent assay. mRNA expressions of T-bet, GATA-3, FoxP3, and RORγt in CD4(+)T cells and perforin and granzyme B in CD8(+)T cells were semi-quantified by real-time PCR. Student t test or paired t test was used for comparisons between the two groups. Results: TIM-3(+) CD4(+) T cell percentage significantly increased in CHF group than that of HC group (3.47%±1.06% vs 0.92%±0.27%, P<0.001). TIM-3(+)CD8(+)T cell percentage also notably elevated in CHF group compared to HC group (6.12%±1.91% vs 1.77%±0.63%, P<0.001). CD4(+)T and CD8(+)T cells were dysfunctional in chronic heart failure. The levels of IFN-γ, IL-17, and IL-22 secreted by purified CD4(+) T cells significantly reduced in CHF group, while IL-10 and IL-35 expressions elevated in CHF group (all P<0.05). The relative mRNA expression levels of T-bet and RORγt in CD4(+) T cells remarkably decreased in CHF group than those of HC group (all P<0.01), while relative expression level of FoxP3 mRNA increased in CHF group (1.93±0.88 vs 0.97±0.28, P=0.031). The levels of TNF-α and IFN-γ produced by purified CD8(+)T cells notably reduced in CHF group than those of HC group (all P<0.05), and relative mRNA expression levels of perforin and granzyme B also decreased in CHF group (all P<0.05). The levels of anti-TIM-3 antibody stimulation-produced IFN-γ, IL-17, and IL-22 increased (all P<0.05) but IL-35 secretion reduced [(61±13) ng/ml vs (72±17) ng/ml, P=0.029] by CD4(+)T cells in CHF group. The relative mRNA expression levels of T-bet and RORγt also elevated in response to anti-TIM-3 antibody stimulation (all P<0.05). Anti-TIM-3 antibody stimulation promoted TNF-α and IFN-γ production by CD8(+)T cells in CHF group (all P<0.01). The relative mRNA expression levels of perforin and granzyme B also increased (all P<0.05). Conclusion: TIM-3 was increasingly expressed in T cells from patients with chronic heart failure, and might take part in the regulation of T cell dysfunction in chronic heart failure.

MSLN (Mesothelin), ANTXR1 (TEM8), and MUC3A are the potent antigenic targets for CAR T cell therapy of gastric adenocarcinoma.

Gastric adenocarcinoma is usually diagnosed in late stages, necessitating the use of different therapeutic modalities. Currently, antibody-based therapies have also been approved through with limited clinical efficacy. Reinforcing antibody-based immunotherapy by using chimeric antigen receptor (CAR) T cells may enhance the approach. However, the cells can cause severe on-target and off-tumor toxicities owing to their higher sensitivity to low-level antigen expressions. To address the need for safe and reliable targets, we made a bioinformatics pipeline by which we screened overexpressed genes in the disease for off-tumor sites in many normal tissues. Our inspection showed that MSLN (Mesothelin), ANTXR1 (TEM8), and MUC3A are the probable targets of CAR T cell therapy in gastric adenocarcinoma. The proposed antigenic targets might respond to the need to simultaneously target multiple antigens in a tumor matrix to prevent resistance.

Multiple Inhibitory Pathways Contribute to Lung CD8+ T Cell Impairment and Protect against Immunopathology during Acute Viral Respiratory Infection.

Viruses are frequent causes of lower respiratory infection (LRI). Programmed cell death-1 (PD-1) signaling contributes to pulmonary CD8(+) T cell (TCD8) functional impairment during acute viral LRI, but the role of TCD8 impairment in viral clearance and immunopathology is unclear. We now find that human metapneumovirus infection induces virus-specific lung TCD8 that fail to produce effector cytokines or degranulate late postinfection, with minimally increased function even in the absence of PD-1 signaling. Impaired lung TCD8 upregulated multiple inhibitory receptors, including PD-1, lymphocyte activation gene 3 (LAG-3), T cell Ig mucin 3, and 2B4. Moreover, coexpression of these receptors continued to increase even after viral clearance, with most virus-specific lung TCD8 expressing three or more inhibitory receptors on day 14 postinfection. Viral infection also increased expression of inhibitory ligands by both airway epithelial cells and APCs, further establishing an inhibitory environment. In vitro Ab blockade revealed that multiple inhibitory receptors contribute to TCD8 impairment induced by either human metapneumovirus or influenza virus infection. In vivo blockade of T cell Ig mucin 3 signaling failed to enhance TCD8 function or reduce viral titers. However, blockade of LAG-3 in PD-1-deficient mice restored TCD8 effector functions but increased lung pathology, indicating that LAG-3 mediates lung TCD8 impairment in vivo and contributes to protection from immunopathology during viral clearance. These results demonstrate that an orchestrated network of pathways modifies lung TCD8 functionality during viral LRI, with PD-1 and LAG-3 serving prominent roles. Lung TCD8 impairment may prevent immunopathology but also contributes to recurrent lung infections.

[Expression and distribution of programmed death receptor 1 and T cell immunoglobulin mucin 3 in breast cancer microenvironment and its relationship with clinicopathological features].

Objective: To explore the expression and distribution of programmed death receptor 1 (PD-1) and T-cell immunoglobulin mucin 3 (TIM-3) in breast cancer microenvironment and analyze the their correlation with the clinicopathological features. Methods: The specimens of tumor tissue and adjacent tissues from 30 patients with infiltrative breast cancer who were diagnosed as breast cancer from June 2016 to May 2017 in The First Hospital of Jiaxing were collected, and the specimen were divided into two parts along the center. After embedding and cryosectioning, the expression and distribution of PD-1 and TIM-3 protein in tumor tissues were observed by immunofluorescence staining. Another part of the specimen was cut and digested, and non-continuous density gradient centrifugation was used to extract tumor-infiltrating lymphocytes (TILs), real-time quantitative PCR (qRT-PCR) was used to detect the mRNA expression of PD-1 and TIM-3 in TILs. Meanwhile, the protein expression was determined by Western blotting. The relationship between the expression of PD-1 and TIM-3 and pathological parameters of breast cancer was analyzed with correlation analysis. Results: Immunofluorescence results showed that more PD-1 and TIM-3 positive cells were observed in the tumor tissues compared with the tumor-adjacent tissues. The qRT-PCR showed that the expression of PD-1 and TIM-3 mRNA in TILs were both significantly higher than those in paracancerous tissues (3.09±0.38 vs 1.26±0.23, 3.42±0.31 vs 1.57±0.29, t=4.16, 4.37, both P<0.05). At the protein level, the expression of PD-1 and TIM-3 in tumor tissue lymphocytes(0.66±0.08, 0.80±0.11) was significantly higher than those in cancerous tissues(0.10±0.01, 0.26±0.02) (t=6.79, 4.57, both P<0.05). There were significant differences in the expression of PD-1, TIM-3 mRNA in the TILs between the different tumor histological grades, tumor sizes, lymph node metastasis (t=2.22-2.99, all P<0.05). Correlation analysis showed that there was a significant positive correlation between the expression of PD-1 and TIM-3 in tumor tissues (r=0.616, P<0.01). Conclusions: In the breast cancer microenvironment, PD-1, TIM-3-mediated signaling pathway plays an important role in the occurrence and development of breast cancer, it provides a new basis for the combination therapy of breast cancer.

Non-Invasive Evaluation of Extracellular Matrix Formation in the Intestinal Epithelium.

Differentiation of colorectal cancer Caco-2 cells was assessed using Affymetrix Human Gene 1.0 ST arrays and by the main electrical parameters measured by bioimpedance spectroscopy. Transepithelial electrical resistance (TEER) was maximum on day 7, then decreased by day 11, and remained stable. The baseline resistance was maximum on day 4, minimum on day 7, but then gradually increased over 2 weeks, which can be explained by the formation of the basement membrane components or the apical mucous layer. Caco-2 cells express components of laminin-111 and laminin-511. A synchronous increase in the expression of mucin 3 mRNA (MUC3A/MUC3B) and mucin 17 mRNA (MUC17) and reduced expression of miR-21 and miR-622 microRNA genes were observed. Possible use of the described approach for studying the formation of extracellular matrix is discussed.

Phosphatidylcholine passes through lateral tight junctions for paracellular transport to the apical side of the polarized intestinal tumor cell-line CaCo2.

Phosphatidylcholine (PC) is the most abundant phospholipid in intestinal mucus, indicative of a specific transport system across the mucosal epithelium to the intestinal lumen. To elucidate this transport mechanism, we employed a transwell tissue culture system with polarized CaCo2 cells. It was shown that PC could not substantially be internalized by the cells. However, after basal application of increasing PC concentrations, an apical transport of 47.1±6.3nmolh(-1)mMPC(-1) was observed. Equilibrium distribution studies with PC applied in equal concentrations to the basal and apical compartments showed a 1.5-fold accumulation on the expense of basal PC. Disruption of tight junctions (TJ) by acetaldehyde or PPARγ inhibitors or by treatment with siRNA to TJ proteins suppressed paracellular transport by at least 50%. Transport was specific for the choline containing the phospholipids PC, lysoPC and sphingomyelin. We showed that translocation is driven by an electrochemical gradient generated by apical accumulation of Cl(-) and HCO3(-) through CFTR. Pretreatment with siRNA to mucin 3 which anchors in the apical plasma membrane of mucosal cells inhibited the final step of luminal PC secretion. PC accumulates in intestinal mucus using a paracellular, apically directed transport route across TJs.

[Association of Crohn's disease with T cell immunoglobulin and mucin domain 3 gene polymorphisms in Chinese patients].

Objective: To explore the association of Crohn's disease (CD) with T cell immunoglobulin and mucin domain 3 (Tim-3) gene polymorphisms in patients of Zhejiang Han population in China. Methods: A total of 308 CD patients and 573 age- and sex-matched healthy controls were enrolled in our study. Two single nucleotide polymorphisms (SNPs) of Tim-3 (rs1036199 and rs10515746) were examined by the improved multiple ligase detection reaction technique (iMLDR). Analyses of linkage disequilibrium and haplotype were also performed by Haploview 4.2 software in all study subjects. Results: In general, the allele and genotype frequencies of Tim-3 (rs1036199 and rs10515746) were not statistically different between CD patients and the controls (all P>0.05). According to "the Montreal Classification" , CD patients were divided into different subgroups. The variant allele (C) and genotype (AC+ CC) of rs1036199 were more frequent in CD patients with penetrating diseases than in the controls (10.4% vs 1.7%, P=0.002; 20.8% vs 3.5%, P=0.023). Similar conclusions were also drawn for the variant allele (A) and genotype (CA+ AA) of rs10515746 in patients with penetrating diseases when compared with the controls (10.4% vs 2.2%, P=0.000; 20.8% vs 4.2%, P=0.033, respectively). The two SNPs of Tim-3 were in strong linkage disequilibrium (D'=1.0, r2=0.928). The haplotype (AC) formed by their wild-type alleles (A) and (C) was decreased in patients with penetrating CD compared with the controls (89.6% vs 98.3%, P=0.000). However, the haplotype (CA) formed by their variant alleles was more frequent in patients with penetrating CD than in the controls (10.4% vs 1.6%, P=0.000). Conclusions:Tim-3 (rs1036199 and rs10515746) variations might be correlated with the enhanced risk of penetrating diseases in CD patients. Furthermore, the haplotype (AC) and (CA) formed by the two SNPs might be a protective and a risky factor for penetrating CD respectively.

Use of Atomic Force Microscopy to Study the Multi-Modular Interaction of Bacterial Adhesins to Mucins.

The mucus layer covering the gastrointestinal (GI) epithelium is critical in selecting and maintaining homeostatic interactions with our gut bacteria. However, the molecular details of these interactions are not well understood. Here, we provide mechanistic insights into the adhesion properties of the canonical mucus-binding protein (MUB), a large multi-repeat cell-surface adhesin found in Lactobacillus inhabiting the GI tract. We used atomic force microscopy to unravel the mechanism driving MUB-mediated adhesion to mucins. Using single-molecule force spectroscopy we showed that MUB displayed remarkable adhesive properties favouring a nanospring-like adhesion model between MUB and mucin mediated by unfolding of the multiple repeats constituting the adhesin. We obtained direct evidence for MUB self-interaction; MUB-MUB followed a similar binding pattern, confirming that MUB modular structure mediated such mechanism. This was in marked contrast with the mucin adhesion behaviour presented by Galectin-3 (Gal-3), a mammalian lectin characterised by a single carbohydrate binding domain (CRD). The binding mechanisms reported here perfectly match the particular structural organization of MUB, which maximizes interactions with the mucin glycan receptors through its long and linear multi-repeat structure, potentiating the retention of bacteria within the outer mucus layer.

[Association between chronicity of HBV infection and host immunity].

The prognosis of hepatitis B virus (HBV) infection is determined by innate immunity, adaptive immunity, and a variety of regulatory factors in the host. Controversy still exists over the role of innate immunity in the progression of HBV infection. Adaptive immunity, especially the immune response mediated by CD8+ T cells, plays an important role in HBV clearance. However, in patients with chronic infection, such CD8+ T cells are often exhausted and associated with various regulatory factors including programmed cell death 1 and T-cell immunoglobulin mucin-3. This article elaborates on the association of chronicity of HBV infection with host immune system and various regulating factors.

Liver-primed CD8+ T cells suppress antiviral adaptive immunity through galectin-9-independent T-cell immunoglobulin and mucin 3 engagement of high-mobility group box 1 in mice.

UNLABELLED: The liver is a tolerogenic environment exploited by persistent infections, such as hepatitis B (HBV) and C (HCV) viruses. In a murine model of intravenous hepatotropic adenovirus infection, liver-primed antiviral CD8(+) T cells fail to produce proinflammatory cytokines and do not display cytolytic activity characteristic of effector CD8(+) T cells generated by infection at an extrahepatic, that is, subcutaneous, site. Importantly, liver-generated CD8(+) T cells also appear to have a T-regulatory (Treg) cell function exemplified by their ability to limit proliferation of antigen-specific T-effector (Teff ) cells in vitro and in vivo via T-cell immunoglobulin and mucin 3 (Tim-3) expressed by the CD8(+) Treg cells. Regulatory activity did not require recognition of the canonical Tim-3 ligand, galectin-9, but was dependent on CD8(+) Treg cell-surface Tim-3 binding to the alarmin, high-mobility group box 1 (HMGB-1). CONCLUSION: Virus-specific Tim-3(+) CD8(+) T cells operating through HMGB-1 recognition in the setting of acute and chronic viral infections of the liver may act to dampen hepatic T-cell responses in the liver microenvironment and, as a consequence, limit immune-mediated tissue injury or promote the establishment of persistent infections.

Biophysical analysis of the molecular interactions between polysaccharides and mucin.

Mucoadhesive materials adhere persistently to mucosal surfaces. A mucoadhesive delivery system could therefore facilitate the controlled release of drugs and optimize their bioavailability in mucosal tissues. Polysaccharides are the most versatile class of natural polymers for transmucosal drug delivery. We used microviscosimetry to explore the mucoadhesion of a library of polysaccharide families with diverse structural characteristics as a first step toward the rational design of mucoadhesive polysaccharide-based nanoformulations. Here we show that the magnitude of deviation between the viscosity of mixed polysaccharide-mucin solutions and the corresponding individual stock solutions can indicate underlying molecular interactions. We found that nonlinear monotonic curves predicted a correlation between the magnitude of interaction and the ability of polysaccharide coils to contract in the presence of salt (i.e., chain flexibility). Charge-neutral polysaccharides such as dextran and Streptococcus thermophilus exopolysaccharide did not interact with mucin. Synchrotron small-angle X-ray scattering (SAXS) data supported the previously described structural features of mucin. Furthermore, high-q scattering data (i.e., sensitive to smaller scales) revealed that when mucin is in dilute solution (presumably in an extended conformation) in the presence of low-Mw alginate, its structure resembles that observed at higher concentrations in the absence of alginate. This effect was less pronounced in the case of high-Mw alginate, but the latter influenced the bulk properties of mucin-alginate mixtures (e.g., hydrodynamic radius and relative viscosity) more prominently than its low-Mw counterpart.

Activity of ERK regulates mucin 3 expression and is involved in undifferentiated Caco-2 cell death induced by 3-oxo-C12-homoserine lactone.

The signal molecule, 3-oxo-C12-homoserine lactone (3-oxo-C12-HSL), is similar to a mammalian hormone in bacteria. Although most studies have examined the effects of high 3-oxo-C12-HSL concentrations (>200 µM) on mammalian cellular functions because ~600 µM 3-oxo-C12-HSL can be secreted in biofilms of Pseudomonas aeruginosa grown in vitro, we previously showed that a low 3-oxo-C12-HSL concentration (30 µM) induces the apoptosis of undifferentiated Caco-2 cells through suppressing Akt activity. Here, we found that a low concentration of 3-oxo-C12-HSL-activated ERK1/2 in undifferentiated Caco-2 cells. Incubating cells with the ERK pathway inhibitor U0126 for 30 min alleviated the mucin 3 (MUC3) expression suppressed by 3-oxo-C12-HSL, and the upregulation of MUC3 expression induced by a 48-h incubation with U0126-reduced cell death. Thus, altered MUC3 expression caused by long-term attenuated ERK1/2 activity might correlate with the death of undifferentiated Caco-2 cells induced by 3-oxo-C12-HSL.

Effects of epidermal growth factor receptor inhibitor on proliferative cholangitis in hepatolithiasis.

BACKGROUND: There is currently no effective medication to prevent stone recurrence after choledochoscopic lithotomy or to treat proliferative cholangitis (PC), which is the pathologic basis of hepatolithiasis. This study aimed to investigate whether gefitinib, an epidermal growth factor receptor (EGFR) inhibitor, inhibited cholangio hyperplasia and lithogenesis in PC. METHODS: After cholangioscopic lithotomy, indwelling catheters were placed in the diseased bile duct lumens in 94 patients with hepatolithiasis. Subsequently, 49 of the 94 patients were treated with 250 mg gefitinib solution via a catheter twice a week, and they were subjected to choledochoscopic biopsy at 6 and 12 weeks. The rest 45 hepatolithiasis patients without gefitinib treatment served as controls. RESULTS: The expressions of EGFR, PCNA and procollagen I were significantly reduced in the patients treated with gefitinib in 12 weeks compared with those in the control group. Patients in the gefitinib group had a much lower degree of hyperplasia of the biliary epithelium, submucosal glands and collagen fibers compared with those in the control group. Gefitinib treatment significantly decreased mucin 3 expression and beta-glucuronidase activity. CONCLUSION: Postoperative gefitinib treatment could significantly inhibit PC-mediated hyperplasia and lithogenesis, which might provide a novel strategy for the prevention of biliary restenosis and stone recurrence in patients with hepatolithiasis.

Mucin 3 is involved in intestinal epithelial cell apoptosis via N-(3-oxododecanoyl)-L-homoserine lactone-induced suppression of Akt phosphorylation.

N-acyl-homoserine lactones (AHL) are quorum-sensing molecules in bacteria that play important roles in regulating virulence gene expression in pathogens such as Pseudomonas aeruginosa. The present study compared responses between undifferentiated and differentiated Caco-2 cells to N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL). A low concentration of 3-oxo-C12-HSL (30 µM) is sufficient to reduce viability accompanied by apoptosis via the suppression of phosphorylation by Akt in undifferentiated Caco-2 cells. The suppression of Akt phosphorylation appears specific in 3-oxo-C12-HSL, because other AHLs did not influence the phosphorylation status of Akt. The reduced viability induced by 3-oxo-C12-HSL was partially recovered by constitutively active Akt overexpression in undifferentiated Caco-2 cells. Since mucin is considered a vital component of the gut barrier, we investigated whether mucin protects cellular functions induced by 3-oxo-C12-HSL in undifferentiated Caco-2 cells. The results showed that mucin protected undifferentiated Caco-2 cells from apoptosis induced by 3-oxo-C12-HSL. 3-Oxo-C12-HSL did not induce cell death in differentiated Caco-2 cells that expressed higher levels of mucin 3 (MUC3) than undifferentiated Caco-2 cells. In addition, 3-oxo-C12-HSL promoted cell death in undifferentiated Caco-2 cells transfected with MUC3 siRNA and reduced MUC3 expression in undifferentiated Caco-2 cells. Therefore, MUC3 might be responsible for the survival of undifferentiated intestinal epithelial cells in the presence of 3-oxo-C12-HSL through regulating Akt phosphorylation. In conclusion, 3-oxo-C12-HSL might influence the survival of undifferentiated intestinal epithelial cells as well as interactions between these cells and pathogens.