Group 3 innate lymphoid cells produce the growth factor HB-EGF to protect the intestine from TNF-mediated inflammation.

Tumor necrosis factor (TNF) drives chronic inflammation and cell death in the intestine, and blocking TNF is a therapeutic approach in inflammatory bowel disease (IBD). Despite this knowledge, the pathways that protect the intestine from TNF are incompletely understood. Here we demonstrate that group 3 innate lymphoid cells (ILC3s) protect the intestinal epithelium from TNF-induced cell death. This occurs independent of interleukin-22 (IL-22), and we identify that ILC3s are a dominant source of heparin-binding epidermal growth factor-like growth factor (HB-EGF). ILC3s produce HB-EGF in response to prostaglandin E2 (PGE2) and engagement of the EP2 receptor. Mice lacking ILC3-derived HB-EGF exhibit increased susceptibility to TNF-mediated epithelial cell death and experimental intestinal inflammation. Finally, human ILC3s produce HB-EGF and are reduced from the inflamed intestine. These results define an essential role for ILC3-derived HB-EGF in protecting the intestine from TNF and indicate that disruption of this pathway contributes to IBD.

Spread of genetically similar noroviruses in Bangkok, Thailand, through symptomatic and asymptomatic individuals.

Norovirus infection is a major cause of acute gastroenteritis, although some infected individuals are asymptomatic. GII.4 is the predominant genotype worldwide and, since 2000, has been the most prevalent in patients in Thailand with acute gastroenteritis. We screened stool samples for norovirus in 786 patients with acute gastroenteritis who were admitted to a hospital in Bangkok from 2017 to early 2019 and detected it in 136 specimens (17.3%). Eight and 124 specimens were positive for the GI and GII genogroups, respectively, and the remaining 4 specimens were double-positive. Nine genotypes (GI.3, GI.5, GII.2, GII.3, GII.4, GII.6, GII.8, GII.13, and GII.17) were identified from 140 strains, and 72 strains (51.4%) were GII.4. We had previously conducted a one-year survey of norovirus infection in residents of a community in Bangkok from May 2018 to April 2019 and found that a substantial portion of the residents were infected asymptomatically. The 9 genotypes identified in the patients were also commonly identified in the community residents. To investigate the relationship between noroviruses identified in the acute gastroenteritis patients and those identified in the community residents, phylogenetic tree analysis was conducted. Of the 9 genotypes, 8 showed similarities in both their genomic sequences and their deduced amino acid sequences. In addition, strain replacement of GI.3 was observed in both the patients and the community residents within the overlapping period. These results suggested that norovirus spreads efficiently to the community by simultaneously causing symptomatic and asymptomatic infections.

Identification of GII.14[P7] norovirus and its genomic mutations from a case of long-term infection in a post-symptomatic individual.

Norovirus is a leading cause of acute gastroenteritis worldwide. Norovirus shedding typically lasts one week to one month after the onset of diarrhea in immunocompetent hosts. The occurrence of mutations in the genome during infection has contributed to the evolution of norovirus. It has been suggested that genomic mutations in the P2-domain of capsid protein VP1, the major antigenic site for virus clearance, are involved in the evasion of host immunity and prolonged shedding of norovirus. In our previous study, we found a case of long-term shedding of GII.14 norovirus in a post-symptomatic immunocompetent individual that lasted about three months. In this study, we characterized the genomic sequence of the GII.14 strain to gain insight into the context of long-term shedding. By sequencing a 4.8 kb region of the genome corresponding to half of ORF1 and the entire ORF2 and ORF3, which encode several non-structural proteins and the structural proteins VP1 and VP2, the GII.14 strain was found to be classified as recombinant GII.14[P7]. Six point-mutations occurred during the three-month period of infection in a time-dependent manner in the genomic regions encoding RNA-dependent RNA polymerase, VP1, and VP2. Three of the six mutations were sense mutations, but no amino acid substitution was identified in the P2-domain of VP1. These results suggest that there is a mechanism by which long-term shedding of norovirus occurs in immunocompetent individuals independent of P2-domain mutations.

Norovirus transmission mediated by asymptomatic family members in households.

The transmission of human norovirus excreted from infected persons occasionally causes sporadic infections and outbreaks. Both symptomatic patients and asymptomatic carriers have been reported to contribute to norovirus transmission, but little is known about the magnitude of the contribution of asymptomatic carriers. We carried out a 1-year survey of residents of a district of Bangkok, Thailand to determine the percentage of norovirus transmissions originating from asymptomatic individuals. We screened 38 individuals recruited from 16 families from May 2018 to April 2019 for GI and GII genotypes. Norovirus was detected every month, and 101 of 716 stool samples (14.1%) from individuals with no symptoms of acute gastroenteritis were norovirus-positive. The average infection frequency was 2.4 times per person per year. Fourteen genotypes were identified from the positive samples, with GII.4 being detected most frequently. Notably, 89.1% of the norovirus-positive samples were provided by individuals with no diarrhea episode. Similar to cases of symptomatic infections in Thailand, asymptomatic infections were observed most frequently in December. We detected 4 cases of NV infection caused by household transmission, and 3 of the 4 transmissions originated from asymptomatic individuals. We also identified a case in which norovirus derived from an asymptomatic individual caused diarrhea in a family member. These results suggest that asymptomatic individuals play a substantial role in both the maintenance and spreading of norovirus in a community through household transmission.

The use of green fluorescent protein-tagged virus-like particles as a tracer in the early phase of chikungunya infection.

To visually examine the early phase of chikungunya virus (CHIKV) infection in target cells, we constructed a virus-like particle (VLP) in which the envelope protein E1 is fused with green fluorescent protein (GFP). This chikungunya VLP-GFP (CHIK-VLP-EGFP), purified by density gradient fractionation, was observed as 60-70 nm-dia. particles and was detected as tiny puncta of fluorescence in the cells. CHIK-VLP-EGFP showed binding properties similar to those of the wild-type viruses. Most of the fluorescence signals that had bound on Vero cells disappeared within 30 min at 37 °C, but not in the presence of anti-CHIKV neutralizing serum or an endosomal acidification inhibitor (bafilomycin A1), suggesting that the loss of fluorescence signals is due to the disassembly of the viral envelope following the internalization of CHIK-VLP-EGFP. In addition to these results, the fluorescence signals disappeared in highly susceptible Vero and U251MG cells but not in poorly susceptible A549 cells. Thus, CHIK-VLP-EGFP is a useful tool to examine the effects of the CHIKV neutralizing antibodies and antiviral compounds that are effective in the entry phase of CHIKV.

Anti-HB-EGF Antibody-Mediated Delivery of siRNA to Atherosclerotic Lesions in Mice.

For atherosclerotic cardiovascular diseases (ACD), gene therapy may be a potential therapeutic strategy; however, lack of effective and safe methods for gene delivery to atherosclerotic plaques have limited its potential therapeutic applications. To overcome this limitation, we developed a novel antibody-based gene delivery system (anti-HB-EGF/NA vector) by chemically crosslinking antibodies against human heparin-binding epidermal growth factor-like growth factor (HB-EGF). It has been shown to be excessively expressed in human atherosclerotic plaques and NeutrAvidin (NA) for conjugating biotinylated siRNA. Immunofluorescence staining and quantitative flow cytometry analysis using human HB-EGF-expressing cells showed both antibody-mediated selective cellular targeting and efficient intracellular delivery of conjugated biotin-fluorescence. Moreover, we demonstrated antibody-mediated significant and selective gene knockdown via conjugation with anti-HB-EGF/NA vector and biotinylated siRNA (anti-HB-EGF/NA/b-siRNA) in vitro. Furthermore, using high fat-fed human HB-EGF knock-in and apolipoprotein E-knockout (Hbegf hz/hz; Apoe-/-) mice, we demonstrated that the anti-HB-EGF/NA vector, conjugating biotin-fluorescence, increasingly accumulated within the atherosclerotic plaques of the ascending aorta in which human HB-EGF expression levels were highly elevated. Moreover, in response to a single intravenous injection of anti-HB-EGF/NA/b-siRNA in a dose-dependent manner, qPCR analysis of laser-dissected atherosclerotic plaques of the ascending aorta showed significant knockdown of the reporter gene expression. These results suggest that the anti-HB-EGF antibody-mediated siRNA delivery could be a promising delivery system for gene therapy of ACD.

Double deletion of tetraspanins CD9 and CD81 in mice leads to a syndrome resembling accelerated aging.

Chronic obstructive pulmonary disease (COPD) has been recently characterized as a disease of accelerated lung aging, but the mechanism remains unclear. Tetraspanins have emerged as key players in malignancy and inflammatory diseases. Here, we found that CD9/CD81 double knockout (DKO) mice with a COPD-like phenotype progressively developed a syndrome resembling human aging, including cataracts, hair loss, and atrophy of various organs, including thymus, muscle, and testis, resulting in shorter survival than wild-type (WT) mice. Consistent with this, DNA microarray analysis of DKO mouse lungs revealed differential expression of genes involved in cell death, inflammation, and the sirtuin-1 (SIRT1) pathway. Accordingly, expression of SIRT1 was reduced in DKO mouse lungs. Importantly, siRNA knockdown of CD9 and CD81 in lung epithelial cells additively decreased SIRT1 and Foxo3a expression, but reciprocally upregulated the expression of p21 and p53, leading to reduced cell proliferation and elevated apoptosis. Furthermore, deletion of these tetraspanins increased the expression of pro-inflammatory genes and IL-8. Hence, CD9 and CD81 might coordinately prevent senescence and inflammation, partly by maintaining SIRT1 expression. Altogether, CD9/CD81 DKO mice represent a novel model for both COPD and accelerated senescence.

BK-UM in patients with recurrent ovarian cancer or peritoneal cancer: a first-in-human phase-I study.

BACKGROUND: BK-UM (CRM197) is a mutant form of diphtheria toxin and a specific inhibitor of heparin-binding epidermal growth factor-like growth factor (HB-EGF). We assessed the safety, pharmacokinetics, recommended dose, and efficacy of BK-UM in patients with recurrent ovarian cancer (OC) or peritoneal cancer (PC), and measured HB-EGF levels in serum and abdominal fluid after BK-UM administration. METHODS: Eleven patients with advanced or recurrent OC or PC were enrolled and treated with BK-UM via the intraperitoneal route. The dose was escalated (1.0, 2.0, 3.3, and 5.0 mg/m2) using a 3 + 3 design. RESULTS: Eight of 11 patients completed treatment. No dose-limiting toxicity (DLT) was experienced at dose levels 1 (1.0 mg/m2) and 2 (2.0 mg/m2). Grade 3 transient hypotension as an adverse event (defined as a DLT in the present study) was observed in two of four patients at dose level 3 (3.3 mg/m2). Treatment with BK-UM was associated with decreases in HB-EGF levels in serum and abdominal fluid in seven of 11 patients and five of eight patients, respectively. Clinical outcomes included a partial response in one patient, stable disease in five patients, and progressive disease in five patients. CONCLUSIONS: BK-UM was well tolerated at doses of 1.0 and 2.0 mg/m2, with evidence for clinical efficacy in patients with recurrent OC or PC. A dose of 2.0 mg/m2 BK-UM is recommended for subsequent clinical trials. TRIAL REGISTRATION: This trial was prospectively performed as an investigator-initiated clinical trial. The trial numbers are UMIN000001002 and UMIN000001001, with registration dates of 1/30/2008 and 2/4/2008, respectively. UMIN000001001 was registered as a trial for the continuous administration of BK-UM after UMIN000001002 .

ErbB1 and ErbB4 generate opposing signals regulating mesenchymal cell proliferation during valvulogenesis.

Heparin-binding EGF-like growth factor (HB-EGF) plays an indispensable role in suppression of cell proliferation during mouse valvulogenesis. However, ligands of the EGF receptor (EGFR/ErbB1), including HB-EGF, are generally considered as growth-promoting factors, as shown in cancers. HB-EGF binds to and activates ErbB1 and ErbB4. We investigated the role of ErbB receptors in valvulogenesis in vivo using ErbB1- and ErbB4-deficient mice, and an ex vivo model of endocardial cushion explants. We show that HB-EGF suppresses valve mesenchymal cell proliferation through a heterodimer of ErbB1 and ErbB4, and an ErbB1 ligand (or ligands) promotes cell proliferation through a homodimer of ErbB1. Moreover, a rescue experiment with cleavable or uncleavable isoforms of ErbB4 in ERBB4-null cells indicates that the cleavable JM-A, but not the uncleavable JM-B, splice variant of ErbB4 rescues the defect of the null cells. These data suggest that the cytoplasmic intracellular domain of ErbB4, rather than the membrane-anchored tyrosine kinase, achieves this suppression. Our study demonstrates that opposing signals generated by different ErbB dimer combinations function in the same cardiac cushion mesenchymal cells for proper cardiac valve formation.

Reconstitution of a metastatic-resistant tumor microenvironment with cancer-associated fibroblasts enables metastasis.

The tumor microenvironment is critical for metastasis to occur. Subcutaneous xenografts of tumors in immunodeficient mice are usually encapsulated and rarely metastasize as opposed to orthotopic tumors which metastasize if the original tumor was metastatic. In the present report, we were able to reconstitute a metastatic tumor microenvironment by subcutaneously co-transplanting a human cervical cancer cell line and human cervical cancer-associated fibroblasts (CAFs), in athymic mice, which resulted in lymph node metastasis in 40% of the animals. In contrast, no metastasis occurred from the cervical cancer without CAFs. These results suggest that CAFs can overcome an anti-metastatic tumor environment and are a potential target to prevent metastasis.

Characterization of a Novel Anti-Human HB-EGF Monoclonal Antibody Applicable for Paraffin-Embedded Tissues and Diagnosis of HB-EGF-Related Cancers.

Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors that bind to and activate the EGF receptor (EGFR/ErbB1) and ErbB4. HB-EGF plays pivotal roles in pathophysiological processes, including cancer. Thus, monoclonal antibodies (mAbs) for HB-EGF detection could be an important tool in the therapeutic diagnosis of HB-EGF-related cancers and other diseases. However, few mAbs, especially those applicable for immunohistochemistry (IHC), have been established to date. In this study, we generated a clone of hybridoma-derived mAb 2-108 by immunizing mice with recombinant human HB-EGF protein expressed by human cells. The mAb 2-108 specifically bound to human HB-EGF but not to mouse HB-EGF and was successful in immunoblotting, even under reducing conditions, immunoprecipitation, and immunofluorescence for unfixed as well as paraformaldehyde-fixed cells. Notably, this mAb was effective in IHC of paraffin-embedded tumor specimens. Epitope mapping analysis showed that mAb 2-108 recognized the N-terminal prodomain in HB-EGF. These results indicate that this new anti-HB-EGF mAb 2-108 would be useful in the diagnosis of HB-EGF-related cancers and would be a strong tool in both basic and clinical research on HB-EGF.

Identification of diphtheria toxin R domain mutants with enhanced inhibitory activity against HB-EGF.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a ligand of EGF receptor, is involved in the growth and malignant progression of cancers. Cross-reacting material 197, CRM197, a non-toxic mutant of diphtheria toxin (DT), specifically binds to the EGF-like domain of HB-EGF and inhibits its mitogenic activity, thus CRM197 is currently under evaluation in clinical trials for cancer therapy. To develop more potent DT mutants than CRM197, we screened various mutant proteins of R domain of DT, the binding site for HB-EGF. A variety of R-domain mutant proteins fused with maltose-binding protein were produced and their inhibitory activity was evaluated in vitro. We found four R domain mutants that showed much higher inhibitory activity against HB-EGF than wild-type (WT) R domain. These R domain mutants suppressed HB-EGF-dependent cell proliferation more effectively than WT R domain. Surface plasmon resonance revealed their higher affinity to HB-EGF than WT R domain. CRM197(R460H) carrying the newly identified mutation showed increased cell proliferation inhibitory activity and affinity to HB-EGF. These results suggest that CRM197(R460H) or other recombinant proteins carrying newly identified mutation(s) in the R domain are potential therapeutics targeting HB-EGF.

Pre-clinical study of BK-UM, a novel inhibitor of HB-EGF, for ovarian cancer therapy.

BACKGROUND/AIM: Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a member of the epidermal growth factor family, is a target for ovarian cancer therapy. The present study investigated the administration schedule of BK-UM, an anticancer agent targeting HB-EGF. MATERIALS AND METHODS: The ovarian cancer cell line, RMG-I, was injected subcutaneously into five-week-old female nude mice. The BK-UM was administered intraperitoneally, using three administration schedules with different doses. The tumor volume was calculated every week. Statistical significance was assessed using the Mann-Whitney U-test. RESULTS: At doses >0.1 mg/kg, BK-UM displayed significant antitumor effects, although the antitumor effects and body weights of mice did not significantly differ by dose or by three different administration schedules. At a dose <0.1 mg/kg, however, BK-UM had little inhibitory effect on tumor growth. CONCLUSION: Daily administration of BK-UM, which has a potentially dose-dependent antitumor effect, may be the optimal schedule for clinical application.

Antibody-modified lipid nanoparticles for selective delivery of siRNA to tumors expressing membrane-anchored form of HB-EGF.

An Fab' antibody against heparin-binding epidermal growth factor-like growth factor (HB-EGF) was applied to achieve advanced tumor-targeted delivery of siRNA. Lipid nanoparticles (LNP) encapsulating siRNA (LNP-siRNA) were prepared, pegylated, and surface modified with Fab' fragments of anti-HB-EGF antibody (αHB-EGF LNP-siRNA). αHB-EGF LNP-siRNA showed high-binding affinity to recombinant human HB-EGF in a Biacore assay. In addition, αHB-EGF LNP-siRNA selectively associated with cells expressing HB-EGF in vitro. Confocal microscopic images showed that siRNA formulated in αHB-EGF LNP-siRNA was efficiently internalized into MDA-MB-231 human breast cancer cells, on which HB-EGF is highly expressed. In addition, siRNA encapsulated in αHB-EGF LNP induced obvious suppression of both target mRNA and protein levels in MDA-MB-231 cells. These results indicate that αHB-EGF LNP have excellent potential to deliver siRNA to target cancer cells, resulting in effective gene silencing.

Co-occurrence of tetraspanin and ROS generators: Conservation in protein cross-linking and other developmental processes.

The nematode exoskeleton, commonly called the cuticle, is a highly structured extracellular matrix mainly composed of collagen. Secreted collagen molecules from the underlying epidermal cells are cross-linked via their tyrosyl residues. Reactive oxygen species (ROS) are required for the cross-linking reaction to produce tyrosyl radicals. The conserved ROS generator enzyme in C. elegans, BLI-3/CeDUOX1, a homolog of dual oxidases (DUOXs), is responsible for production of hydrogen peroxide. The ROS generation system must be properly controlled since ROS are highly reactive molecules that irreversibly inhibit the functions of cellular components such as nucleic acids and proteins. We recently reported that the ROS generation system directed by BLI-3 requires the tetraspanin protein, TSP-15. Herein we outline the process of cuticle development with a focus on the molecular roles of TSP-15 in the BLI-3 system. We also propose the co-occurrence of tetraspanin and ROS generators by convergent evolution.

Metastasis suppressor tetraspanin CD82/KAI1 regulates ubiquitylation of epidermal growth factor receptor.

Ligand-induced ubiquitylation of EGF receptor (EGFR) is an important regulatory mechanism that controls endocytic trafficking of the receptor and its signaling potential. Here we report that tetraspanin CD82/KAI1 specifically suppresses ubiquitylation of EGFR after stimulation with heparin-binding EGF or amphiregulin and alters the rate of recruitment of the activated receptor to EEA1-positive endosomes. The suppressive effect of CD82 is dependent on the heparin-binding domain of the ligand. Deletion of the C-terminal cytoplasmic domain of CD82 (CD82ΔC mutant) inhibits endocytic trafficking of the tetraspanin and compromises its activity toward heparin-binding EGF-activated EGFR. Reduced ubiquitylation of EGFR is accompanied by PKC-dependent increase in serine phosphorylation of c-Cbl in cells expressing elevated levels of CD82. Furthermore, phosphorylation of threonine 654 (PKC phosphorylation site) in the juxtamembrane domain of the receptor is considerably increased in CD82-expressing cells. These results describe previously unsuspected links between tetraspanin proteins and ubiquitylation of their molecular partners (e.g., EGFR). Our data identify CD82 as a new regulator of c-Cbl, which discriminatively controls the activity of this E3 ubiquitin ligase toward heparin-binding ligand-EGFR pairs. Taken together, these observations provide an important new insight into the modulatory role of CD82 in endocytic trafficking of EGF receptor.

Conditional loss of heparin-binding EGF-like growth factor results in enhanced liver fibrosis after bile duct ligation in mice.

Our aims were to evaluate the involvement of heparin-binding EGF-like growth factor (HB-EGF) in liver fibrogenesis of humans and mice and to elucidate the effect of HB-EGF deficiency on cholestatic liver fibrosis using conditional HB-EGF knockout (KO) mice. We first demonstrated that gene expression of HB-EGF had a positive significant correlation with that of collagen in human fibrotic livers, and was increased in bile duct ligation (BDL)-induced fibrotic livers in mouse. We then generated conditional HB-EGF knockout (KO) mice using the interferon inducible Mx-1 promoter driven Cre recombinase transgene and wild type (WT) and KO mice were subjected to BDL. After BDL, KO mice exhibited enhanced liver fibrosis with increased expression of collagen, compared with WT mice. Finally, we used mouse hepatic stellate cells (HSCs) to examine the role of HB-EGF in the activation of these cells and showed that HB-EGF antagonized TGF-ß-induced gene expression of collagen in mouse primary HSCs. Interestingly, HB-EGF did not prevent the TGF-ß-induced nuclear accumulation of Smad3, but did lead to stabilization of the Smad transcriptional co-repressor TG-interacting factor. In conclusion, our data suggest a possible protective role of HB-EGF in cholestatic liver fibrosis.

Conditional knockout of heparin-binding epidermal growth factor-like growth factor in the liver accelerates carbon tetrachloride-induced liver injury in mice.

AIM: We previously demonstrated that heparin-binding epidermal growth factor-like growth factor (HB-EGF) is induced in response to several liver injuries. Because the HB-EGF knockout (KO) mice die in utero or immediately after birth due to cardiac defects, the loss of function study in vivo is limited. Here, we generated liver-specific HB-EGF conditional knockout mice using the interferon-inducible Mx-1 promoter driven cre recombinase transgene and investigated its role during acute liver injury. METHODS: We induced acute liver injury by a single i.p. injection of carbon tetrachloride (CCl4 ) in HB-EGF KO mice and wild-type mice and liver damage was assessed by biochemical and immunohistochemical analysis. We also used AML12 mouse hepatocyte cell lines to examine the molecular mechanism of HB-EGF-dependent anti-apoptosis and wound-healing process of the liver in vitro. RESULTS: HB-EGF KO mice exhibited a significant increase of alanine aminotransferase level and also showed a significant increase in the number of apoptotic hepatocytes assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling staining at 24 h after CCl4 injection. We also demonstrated that HB-EGF treatment inhibited tumor necrosis factor-α-induced apoptosis of AML12 mouse hepatocytes and promoted the wound-healing response of these cells. CONCLUSION: This study showed that HB-EGF plays a protective role during acute liver injury.

Soluble heparin-binding EGF-like growth factor (HB-EGF) detected by newly developed immuno-PCR method is a clear-cut serological biomarker for ovarian cancer.

Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors that bind to and activate the EGF receptor. HB-EGF is synthesized as a membrane-anchored protein (proHB-EGF), and then proteolytically cleaved, resulting in the mitogenically active soluble form. HB-EGF plays pivotal roles in pathophysiological processes such as development and cell proliferation. In this study, we developed an immuno-PCR system for the determination of soluble HB-EGF concentrations in human serum. Utilizing a monoclonal antibody with neutralizing activity against HB-EGF as a capture antibody resulted in increasing selectivity for an active form of HB-EGF in serum, and immuno-PCR system improved its sensitivity compared to the currently available methods. As a result of measurement of HB-EGF in 20 ovarian cancer patients and 20 healthy volunteers, ovarian cancer patients showed significantly higher concentrations than healthy volunteers (P< 0.05), which indicates that soluble HB-EGF detected by newly developed immuno-PCR system can be useful serological biomarkers such as a diagnostic biomarker for ovarian cancer, and a predictive and pharmacodynamic biomarker for anti-HB-EGF targeted therapies under development.