Construction of a Lactobacillus plantarum-based claudin-3 targeting delivery system for the development of vaccines against Eimeria tenella.

Avian coccidiosis causes huge economic losses to the poultry industry worldwide and currently lacks effective live vector vaccines. Achieving efficient antigen delivery to mucosa-associated lymphoid tissue (MALT) is critical for improving the effectiveness of vaccines. Here, chicken claudin-3 (CLDN3), a tight junction protein expressed in MALT, was identified as a target, and the C-terminal region of Clostridium perfringens enterotoxin (C-CPE) was proven to bind to chicken CLDN3. Then, a CLDN3-targeting Lactobacillus plantarum NC8-expressing C-CPE surface display system (NC8/GFP-C-CPE) was constructed to successfully express the heterologous protein on the surface of L. plantarum. The colonization level of NC8/GFP-C-CPE was significantly increased compared to the non-targeting strain and could persist in the intestine for at least 72 h. An oral vaccine strain expressing five EGF domains of Eimeria tenella microneme protein 8 (EtMIC8-EGF) (NC8/EtMIC8-EGF-C-CPE) was constructed to evaluate the protective efficacy against E. tenella infection. The results revealed that CLDN3-targeting L. plantarum induced stronger mucosal immunity in gut-associated lymphoid tissues (GALT) as well as humoral responses and conferred better protection in terms of parasite replication and pathology than the non-targeting strain. Overall, we successfully constructed a CLDN3-targeting L. plantarum NC8 surface display system characterized by MALT-targeting, which is an efficient antigen delivery system to confer enhanced protective efficacy in chickens against E. tenella infection.

Depletion of Intestinal Microbiome Partially Rescues Bone Loss in Sickle Cell Disease Male Mice.

Osteoporosis or osteopenia are common clinical manifestations of sickle cell disease (SCD) with unclear mechanisms. Since senescence of circulating neutrophil can be modulated by signals derived from intestinal microbiome and neutrophils are abundant in bone marrow and can regulate osteoblasts and osteoclasts, we examined whether gut microbiome contributes to bone loss in SCD mice. SCD and their littermates control mice were treated with antibiotics to deplete gut microbiome. At the end of 7 weeks treatment, serum was collected for biochemistry marker measurements. Bone mass and remodeling were evaluated by dual beam X-ray absorptiometry, micro-computed tomography, and histomorphometry. Bone-related genes in tibia and barrier marker genes in the small intestine were analyzed by quantitative PCR. Antibiotic treatment rescued increased intestinal inflammatory cytokine marker genes (Tnfα, IL17, Ifnγ) expression, rescued decreased intestinal barrier marker genes (claudin 3 and claudin 15) expression, and rescued increased serum cytokines (IFNγ, IL27, IL10) in SCD mice. Antibiotic significantly improved decreased bone mass in SCD mice mainly through enhanced osteoblast function and increased osteoblast-related genes (Runx2 and Igf1) expression in SCD mice. Our findings support that increased bacteria load augments antigenic load traversing the impaired intestinal barrier through inflammation, leading to increased inflammatory cytokines, impaired osteoblast function, and bone loss in SCD mice.

Development of Human Monoclonal Antibody for Claudin-3 Overexpressing Carcinoma Targeting.

Most malignant tumors originate from epithelial tissues in which tight junctions mediate cell-cell interactions. Tight junction proteins, especially claudin-3 (CLDN3), are overexpressed in various cancers. Claudin-3 is exposed externally during tumorigenesis making it a potential biomarker and therapeutic target. However, the development of antibodies against specific CLDN proteins is difficult, because CLDNs are four-transmembrane domain proteins with high homology among CLDN family members and species. Here, we developed a human IgG1 monoclonal antibody (h4G3) against CLDN3 through scFv phage display using CLDN3-overexpressing stable cells and CLDN3-embedded lipoparticles as antigens. The h4G3 recognized the native conformation of human and mouse CLDN3 without cross-reactivity to other CLDNs. The binding kinetics of h4G3 demonstrated a sub-nanomolar affinity for CLDN3 expressed on the cell surface. The h4G3 showed antibody-dependent cellular cytotoxicity (ADCC) according to CLDN3 expression levels in various cancer cells by the activation of FcγRIIIa (CD16a). The biodistribution of h4G3 was analyzed by intravenous injection of fluorescence-conjugated h4G3 which showed that it localized to the tumor site in xenograft mice bearing CLDN3-expressing tumors. These results indicate that h4G3 recognizes CLDN3 specifically, suggesting its value for cancer diagnosis, antibody-drug conjugates, and potentially as a chimeric antigen receptor (CAR) for CLDN3-expressing pan-carcinoma.

Probiotic Bacillus enhance the intestinal epithelial cell barrier and immune function of piglets.

Bacillus is widely used in the livestock industry. This study was designed to evaluate the effects of probiotic Bacillus amyloliquefaciens SC06 (Ba), originally isolated from soil, in piglets diet as an alternative to antibiotics (aureomycin), mainly on intestinal epithelial barrier and immune function. Ninety piglets were divided into three groups: G1 (containing 150 mg/kg aureomycin in the diet); G2 (containing 75 mg/kg aureomycin and 1×108 cfu/kg Ba in the diet); G3 (containing 2×108 cfu/kg Ba in the diet without any antibiotics). The results showed that, compared with the antibiotic group (G1), villus length, crypt depth and villus length/crypt depth ratio of intestine significantly increased in the G2 and G3 groups. In addition, intestinal villi morphology, goblet-cell number, mitochondria structure and tight junction proteins of intestinal epithelial cells in G2 and G3 were better than in G1. The relative gene expression of intestinal mucosal defensin-1, claudin3, claudin4, and human mucin-1 in G3 was significantly lower, while the expression of villin was significantly higher than in the antibiotic group. Probiotic Ba could significantly decrease serum interferon (IFN)-α, IFN-γ, interleukin (IL)-1ß, and IL-4 levels, whereas increase tumour necrosis factor (TNF)-α and IL-6 secretion. Ba could also significantly decrease cytokines TNF-α, IFN-γ, IL-1ß, and IL-4 level in liver, whereas it significantly increased IFN-α. Furthermore, replacing antibiotics with Ba also significantly down-regulated gene expression of TNF and IL-1α in intestinal mucosa, but up-regulated IL-6 and IL-8 transcription. Dietary addition of Ba could significantly reduce the gene expression of nuclear factor kappa beta (NFκB)-p50 and Toll-like receptor (TLR)6, while there was no significant difference for that of myeloid differentiation primary response 88, TNF receptor-associated factor-6, nucleotide-binding oligomerisation domain-containing protein 1, TLR2, TLR4, and TLR9. Taken together, our findings demonstrated that probiotic Ba could increase the intestinal epithelial cell barrier and immune function by improving intestinal mucosa structure, tight junctions and by activating the TLRs signalling pathway.

Blood-brain barrier and intestinal epithelial barrier alterations in autism spectrum disorders.

BACKGROUND: Autism spectrum disorders (ASD) are complex conditions whose pathogenesis may be attributed to gene-environment interactions. There are no definitive mechanisms explaining how environmental triggers can lead to ASD although the involvement of inflammation and immunity has been suggested. Inappropriate antigen trafficking through an impaired intestinal barrier, followed by passage of these antigens or immune-activated complexes through a permissive blood-brain barrier (BBB), can be part of the chain of events leading to these disorders. Our goal was to investigate whether an altered BBB and gut permeability is part of the pathophysiology of ASD. METHODS: Postmortem cerebral cortex and cerebellum tissues from ASD, schizophrenia (SCZ), and healthy subjects (HC) and duodenal biopsies from ASD and HC were analyzed for gene and protein expression profiles. Tight junctions and other key molecules associated with the neurovascular unit integrity and function and neuroinflammation were investigated. RESULTS: Claudin (CLDN)-5 and -12 were increased in the ASD cortex and cerebellum. CLDN-3, tricellulin, and MMP-9 were higher in the ASD cortex. IL-8, tPA, and IBA-1 were downregulated in SCZ cortex; IL-1b was increased in the SCZ cerebellum. Differences between SCZ and ASD were observed for most of the genes analyzed in both brain areas. CLDN-5 protein was increased in ASD cortex and cerebellum, while CLDN-12 appeared reduced in both ASD and SCZ cortexes. In the intestine, 75% of the ASD samples analyzed had reduced expression of barrier-forming TJ components (CLDN-1, OCLN, TRIC), whereas 66% had increased pore-forming CLDNs (CLDN-2, -10, -15) compared to controls. CONCLUSIONS: In the ASD brain, there is an altered expression of genes associated with BBB integrity coupled with increased neuroinflammation and possibly impaired gut barrier integrity. While these findings seem to be specific for ASD, the possibility of more distinct SCZ subgroups should be explored with additional studies.

AIM2 contributes to the maintenance of intestinal integrity via Akt and protects against Salmonella mucosal infection.

The mechanism regulating the gastrointestinal epithelial barrier remains poorly understood. We herein demonstrate that Absent in melanoma-2 (AIM2) contributes to the maintenance of intestinal barrier integrity and defense against bacterial infection. AIM2-deficient mice displayed an increased susceptibility to mucosal but not systemic infection by Salmonella typhimurium, indicating a protective role for AIM2 in the gastrointestinal tract. In a Salmonella colitis model, compared with wild-type mice, AIM2(-/-) mice exhibited more severe body weight loss, intestinal damage, intestinal inflammation, and disruption of basal and activated epithelial cell turnover. In vivo and in vitro data showed that AIM2 restricted the early epithelial paracellular invasion of Salmonella and decreased epithelial permeability. The decreased epithelial barrier in AIM2(-/-) mice might be attributed to the altered expression of tight junction proteins that contribute to epithelial integrity. AIM2 promoted the expression of tight junction proteins through Akt activation. Together, these results suggest that AIM2 is required for maintaining the integrity of the epithelial barrier.

Evaluation of a novel human IgG1 anti-claudin3 antibody that specifically recognizes its aberrantly localized antigen in ovarian cancer cells and that is suitable for selective drug delivery.

Membrane protein claudin3 has been recently suggested as a marker for biologically aggressive tumors and a possible target for the therapeutic delivery of active anti-cancer compounds. Claudin3-binding molecules such as the Clostridium perfringens enterotoxin (CPE), CPE-related molecules, and murine and chimeric antibodies have shown promising antitumor efficacy in preclinical oncological settings. We first engineered a fully human anti-claudin3 IgG1 antibody (IgGH6) by fusing the human IgG1 Fc-domain to the anti-claudin3 scFvH6 previously isolated from a pre-immune phage display library. The construct was expressed in mammalian cells and specifically targeted claudin3 endogenously expressed on the surface of different human ovarian cancer cell lines. No detectable cross-reactivity with other homologous claudins was observed. The epitope recognized by IgGH6 is located within the minor extracellular domain of claudin3 and becomes accessible only in tumor cells characterized by incomplete junction formation. Confocal microscopy experiments demonstrated that IgGH6 was actively internalized in tumor cells after binding to native claudin3 and co-localized, likely within intracellular vesicles, with the C-CPE peptide. Preliminary results indicate that IgGH6 accumulated in vivo in free claudin3 ovarian carcinoma xenografts. For its selective uptake in tumor cells and its human nature, IgGH6 represents a valuable candidate for antibody-drug conjugate therapeutic applications in ovarian cancer patients.

Generation of specific monoclonal antibodies against the extracellular loops of human claudin-3 by immunizing mice with target-expressing cells.

Human claudin-3 (CLDN3) is a tetraspanin transmembrane protein of tight junction structures and is known to be over-expressed in some malignant tumors. Although a specific monoclonal antibody (MAb) against the extracellular domains of CLDN3 would be a valuable tool, generation of such MAbs has been regarded as difficult using traditional hybridoma techniques, because of the conserved sequence homology of CLDN3s among various species. In addition, high sequence similarity is shared among claudin family members, and potential cross-reactivity of MAb should be evaluated carefully. To overcome these difficulties, we generated CLDN3-expressing Chinese hamster ovary and Sf9 cells to use an immunogens and performed cell-based screening to eliminate cross-reactive antibodies. As a result, we generated MAbs that recognized the extracellular loops of CLDN3 but not those of CLDN4, 5, 6, or 9. Further in vitro studies suggested that the isolated MAbs possessed the desired binding properties for the detection or targeting of CLDN3.

Three distinct subsets of thymic epithelial cells in rats and mice defined by novel antibodies.

AIM: Thymic epithelial cells (TECs) are thought to play an essential role in T cell development and have been detected mainly in mice using lectin binding and antibodies to keratins. Our aim in the present study was to create a precise map of rat TECs using antibodies to putative markers and novel monoclonal antibodies (i.e., ED 18/19/21 and anti-CD205 antibodies) and compare it with a map from mouse counterparts and that of rat thymic dendritic cells. RESULTS: Rat TECs were subdivided on the basis of phenotype into three subsets; ED18+ED19+/-keratin 5 (K5)+K8+CD205+ class II MHC (MHCII)+ cortical TECs (cTECs), ED18+ED21-K5-K8+Ulex europaeus lectin 1 (UEA-1)+CD205- medullary TECs (mTEC1s), and ED18+ED21+K5+K8dullUEA-1-CD205- medullary TECs (mTEC2s). Thymic nurse cells were defined in cytosmears as an ED18+ED19+/-K5+K8+ subset of cTECs. mTEC1s preferentially expressed MHCII, claudin-3, claudin-4, and autoimmune regulator (AIRE). Use of ED18 and ED21 antibodies revealed three subsets of TECs in mice as well. We also detected two distinct TEC-free areas in the subcapsular cortex and in the medulla. Rat dendritic cells in the cortex were MHCII+CD103+ but negative for TEC markers, including CD205. Those in the medulla were MHCII+CD103+ and CD205+ cells were found only in the TEC-free area. CONCLUSION: Both rats and mice have three TEC subsets with similar phenotypes that can be identified using known markers and new monoclonal antibodies. These findings will facilitate further analysis of TEC subsets and DCs and help to define their roles in thymic selection and in pathological states such as autoimmune disorders.

Development of an anti-claudin-3 and -4 bispecific monoclonal antibody for cancer diagnosis and therapy.

Most malignant tumors are derived from epithelium, and claudin (CLDN)-3 and CLDN-4 are frequently overexpressed in such tumors. Although antibodies have potential in cancer diagnostics and therapy, development of antibodies against CLDNs has been difficult because the extracellular domains of CLDNs are too small and there is high homology among human, rat, and mouse sequences. Here, we created a monoclonal antibody that recognizes human CLDN-3 and CLDN-4 by immunizing rats with a plasmid vector encoding human CLDN-4. A hybridoma clone that produced a rat monoclonal antibody recognizing both CLDN-3 and -4 (clone 5A5) was obtained from a hybridoma screen by using CLDN-3- and -4-expressing cells; 5A5 did not bind to CLDN-1-, -2-, -5-, -6-, -7-, or -9-expressing cells. Fluorescence-conjugated 5A5 injected into xenograft mice bearing human cancer MKN74 or LoVo cells could visualize the tumor cells. The human-rat chimeric IgG1 monoclonal antibody (xi5A5) activated FcγRIIIa in the presence of CLDN-3- or -4-expressing cells, indicating that xi5A5 may exert antibody-dependent cellular cytotoxicity. Administration of xi5A5 attenuated tumor growth in xenograft mice bearing MKN74 or LoVo cells. These results suggest that 5A5 shows promise in the development of a diagnostic and therapeutic antibody for cancers.

Rapid remodeling of tight junctions during paracellular diapedesis in a human model of the blood-brain barrier.

Leukocyte transendothelial migration (TEM; diapedesis) is a critical event in immune surveillance and inflammation. Most TEM occurs at endothelial cell borders (paracellular). However, there is indirect evidence to suggest that, at the tight junctions of the blood-brain barrier (BBB), leukocytes migrate directly through the endothelial cell body (transcellular). Why leukocytes migrate through the endothelial cell body rather than the cell borders is unknown. To test the hypothesis that the tightness of endothelial cell junctions influences the pathway of diapedesis, we developed an in vitro model of the BBB that possessed 10-fold higher electrical resistance than standard culture conditions and strongly expressed the BBB tight junction proteins claudin-5 and claudin-3. We found that paracellular TEM was still the predominant pathway (≥98%) and TEM was dependent on PECAM-1 and CD99. We show that endothelial tight junctions expressing claudin-5 are dynamic and undergo rapid remodeling during TEM. Membrane from the endothelial lateral border recycling compartment is mobilized to the exact site of tight junction remodeling. This preserves the endothelial barrier by sealing the intercellular gaps with membrane and engaging the migrating leukocyte with unligated adhesion molecules (PECAM-1 and CD99) as it crosses the cell border. These findings provide new insights into leukocyte-endothelial interactions at the BBB and suggest that tight junctions are more dynamic than previously appreciated.

Protein kinase Cß as a therapeutic target stabilizing blood-brain barrier disruption in experimental autoimmune encephalomyelitis.

Disruption of the blood-brain barrier (BBB) is a hallmark of acute inflammatory lesions in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis. This disruption may precede and facilitate the infiltration of encephalitogenic T cells. The signaling events that lead to this BBB disruption are incompletely understood but appear to involve dysregulation of tight-junction proteins such as claudins. Pharmacological interventions aiming at stabilizing the BBB in MS might have therapeutic potential. Here, we show that the orally available small molecule LY-317615, a synthetic bisindolylmaleimide and inhibitor of protein kinase Cß, which is clinically under investigation for the treatment of cancer, suppresses the transmigration of activated T cells through an inflamed endothelial cell barrier, where it leads to the induction of the tight-junction molecules zona occludens-1, claudin 3, and claudin 5 and other pathways critically involved in transendothelial leukocyte migration. Treatment of mice with ongoing experimental autoimmune encephalomyelitis with LY-317615 ameliorates inflammation, demyelination, axonal damage, and clinical symptoms. Although LY-317615 dose-dependently suppresses T-cell proliferation and cytokine production independent of antigen specificity, its therapeutic effect is abrogated in a mouse model requiring pertussis toxin. This abrogation indicates that the anti-inflammatory and clinical efficacy is mainly mediated by stabilization of the BBB, thus suppressing the transmigration of encephalitogenic T cells. Collectively, our data suggest the involvement of endothelial protein kinase Cß in stabilizing the BBB in autoimmune neuroinflammation and imply a therapeutic potential of BBB-targeting agents such as LY-317615 as therapeutic approaches for MS.