Christina Ward

Regulation of Heterotypic Claudin Compatibility

Tissue barrier function is directly mediated by tight junction transmembrane proteins known as claudins. Cells that form tight junctions typically express multiple claudin isoforms which suggests that heterotypic (head-to-head) binding between different claudin isoforms may play a role in regulating paracellular permeability. However, little is known about motifs that control heterotypic claudin compatibility. We found that although claudin-3 and claudin-4 were heteromerically compatible when expressed in the same cell, they did not heterotypically interact despite having extracellular loop (EL) domains that are highly conserved at the amino acid level. Claudin-1 and -5, which were heterotypically compatible with claudin-3, did not heterotypically bind to claudin-4. In contrast, claudin-4 chimeras containing either the first EL domain or the second EL domain of claudin-3 were able to heterotypically bind to claudin-1, claudin-3, and claudin-5. Moreover, a single point mutation in the first extracellular loop domain of claudin-3 to convert Asn44 to the corresponding amino acid in claudin-4 (Thr) produced a claudin capable of heterotypic binding to claudin-4 while still retaining the ability to bind to claudin-1 and -5. Thus, control of heterotypic claudin-claudin interactions is sensitive to small changes in the EL domains.

Differential effects of claudin-3 and claudin-4 on alveolar epithelial barrier function

Alveolar barrier function depends critically on the claudin family tight junction proteins. Of the major claudins expressed by alveolar epithelial cells, claudin (Cldn)-3 and Cldn-4 are the most closely related by amino acid homology, yet they differ dramatically in the pattern of expression. Previously published reports have shown that Cldn-3 is predominantly expressed by type II alveolar epithelial cells; Cldn-4 is expressed throughout the alveolar epithelium and is specifically upregulated in response to acute lung injury. Using primary rat alveolar epithelial cells transduced with yellow fluorescent protein-tagged claudin constructs, we have identified roles for Cldn-3 and Cldn-4 in alveolar epithelial barrier function. Surprisingly, increasing expression of Cldn-3 decreased alveolar epithelial barrier function, as assessed by transepithelial resistance and dye flux measurements. Conversely, increasing Cldn-4 expression improved alveolar epithelial transepithelial resistance compared with control cells. Other alveolar epithelial tight junction proteins were largely unaffected by increased expression of Cldn-3 and Cldn-4. Taken together, these results demonstrate that, in the context of the alveolar epithelium, Cldn-3 and Cldn-4 have different effects on paracellular permeability, despite significant homology in their extracellular loop domains.

Extracellular Matrix Influences Alveolar Epithelial Claudin Expression and Barrier Function

The lung is dynamically remodeled in response to injury, which alters extracellular matrix composition, and can lead to either healthy or impaired lung regeneration. To determine how changes in extracellular matrix can influence alveolar epithelial barrier function, we examined the expression and function of tight junction proteins by rat alveolar epithelial type II cells cultured on one of three different matrix components: type I collagen or fibronectin, matrix glycoproteins which are highly expressed in injured lungs, or laminin, a basement membrane matrix component. Of note, alveolar epithelial cells cultured for 2 days on fibronectin formed high-resistance barriers and showed continuous claudin-3 and claudin-18 localization to the plasma membrane, as opposed to cells cultured on either type I collagen or laminin, which had low resistance monolayers and had areas of cell-cell contact that were claudin deficient. The barrier formed by cells cultured on fibronectin also had preferential permeability to chloride as compared with sodium. Regardless of the initial matrix composition, alveolar epithelial cells cultured for 5 days formed high-resistance barriers, which correlated with increased claudin-18 localization to the plasma membrane and an increase in zonula occludens-1. Day 5 cells on laminin had significantly higher resistance than cells on either fibronectin or type I collagen. Thus, although alveolar epithelial cells on fibronectin formed rapid barriers, it was at the expense of producing an optimized barrier.

Chronic Alcohol Ingestion Increases Mortality and Organ Injury in a Murine Model of Septic Peritonitis

Background Patients admitted to the intensive care unit with alcohol use disorders have increased morbidity and mortality. The purpose of this study was to determine how chronic alcohol ingestion alters the host response to sepsis in mice. Methods Mice were randomized to receive either alcohol or water for 12 weeks and then subjected to cecal ligation and puncture. Mice were sacrificed 24 hours post-operatively or followed seven days for survival. Results Septic alcohol-fed mice had a significantly higher mortality than septic water-fed mice (74% vs. 41%, p = 0.01). This was associated with worsened gut integrity in alcohol-fed mice with elevated intestinal epithelial apoptosis, decreased crypt proliferation and shortened villus length. Further, alcohol-fed mice had higher intestinal permeability with decreased ZO-1 and occludin protein expression in the intestinal tight junction. The frequency of splenic and bone marrow CD4+ T cells was similar between groups; however, splenic CD4+ T cells in septic alcohol-fed mice had a marked increase in both TNF and IFN-γ production following ex vivo stimulation. Neither the frequency nor function of CD8+ T cells differed between alcohol-fed and water-fed septic mice. NK cells were decreased in both the spleen and bone marrow of alcohol-fed septic mice. Pulmonary myeloperoxidase levels and BAL levels of G-CSF and TFG-β were higher in alcohol-fed mice. Pancreatic metabolomics demonstrated increased acetate, adenosine, xanthine, acetoacetate, 3-hydroxybutyrate and betaine in alcohol-fed mice and decreased cytidine, uracil, fumarate, creatine phosphate, creatine, and choline. Serum and peritoneal cytokines were generally similar between alcohol-fed and water-fed mice, and there were no differences in bacteremia, lung wet to dry weight, or pulmonary, liver or splenic histology. Conclusions When subjected to the same septic insult, mice with chronic alcohol ingestion have increased mortality. Alterations in intestinal integrity, the host immune response, and pancreatic metabolomics may help explain this differential response.

Theranostic Nanoparticles Carrying Doxorubicin Attenuate Targeting Ligand Specific Antibody Responses Following Systemic Delivery

Understanding the effects of immune responses on targeted delivery of nanoparticles is important for clinical translations of new cancer imaging and therapeutic nanoparticles. In this study, we found that repeated administrations of magnetic iron oxide nanoparticles (IONPs) conjugated with mouse or human derived targeting ligands induced high levels of ligand specific antibody responses in normal and tumor bearing mice while injections of unconjugated mouse ligands were weakly immunogenic and induced a very low level of antibody response in mice. Mice that received intravenous injections of targeted and polyethylene glycol (PEG)-coated IONPs further increased the ligand specific antibody production due to differential uptake of PEG-coated nanoparticles by macrophages and dendritic cells. However, the production of ligand specific antibodies was markedly inhibited following systemic delivery of theranostic nanoparticles carrying a chemotherapy drug, doxorubicin. Targeted imaging and histological analysis revealed that lack of the ligand specific antibodies led to an increase in intratumoral delivery of targeted nanoparticles. Results of this study support the potential of further development of targeted theranostic nanoparticles for the treatment of human cancers.

NF-κB inhibitors impair lung epithelial tight junctions in the absence of inflammation

NF-κB (p50/p65) is the best characterized transcription factor known to regulate cell responses to inflammation. However, NF-κB is also constitutively expressed. We used inhibitors of the classical NF-κB signaling pathway to determine whether this transcription factor has a role in regulating alveolar epithelial tight junctions. Primary rat type II alveolar epithelial cells were isolated and cultured on Transwell permeable supports coated with collagen for 5 d to generate a model type I cell monolayer. Treatment of alveolar epithelial monolayers overnight with one of 2 different IκB kinase inhibitors (BAY 11–7082 or BMS-345541) resulted in a dose-dependent decrease in TER at concentrations that did not affect cell viability. In response to BMS-345541 treatment there was an increase in total claudin-4 and claudin-5 along with a decrease in claudin-18, as determined by immunoblot. However, there was little effect on the total amount of cell-associated claudin-7, occludin, junctional adhesion molecule A (JAM-A), zonula occludens (ZO)-1 or ZO-2. Moreover, treatment with BMS-345541 resulted in altered tight junction morphology as assessed by immunofluorescence microscopy. Cells treated with BMS-345541 had an increase in claudin-18 containing projections emanating from tight junctions (“spikes”) that were less prominent in control cells. There also were several areas of cell-cell contact which lacked ZO-1 and ZO-2 localization as well as rearrangements to the actin cytoskeleton in response to BMS-345541. Consistent with an anti-inflammatory effect, BMS-345541 antagonized the deleterious effects of lipopolysaccharide (LPS) on alveolar epithelial barrier function. However, BMS-345541 also inhibited the ability of GM-CSF to increase alveolar epithelial TER. These data suggest a dual role for NF-κB in regulating alveolar barrier function and that constitutive NF-κB function is required for the integrity of alveolar epithelial tight junctions.

Junctional Adhesion Molecule A Promotes Epithelial Tight Junction Assembly to Augment Lung Barrier Function

Epithelial barrier function is maintained by tight junction proteins that control paracellular fluid flux. Among these proteins is junctional adhesion molecule A (JAM-A), an Ig fold transmembrane protein. To assess JAM-A function in the lung, we depleted JAM-A in primary alveolar epithelial cells using shRNA. In cultured cells, loss of JAM-A caused an approximately 30% decrease in transepithelial resistance, decreased expression of the tight junction scaffold protein zonula occludens 1, and disrupted junctional localization of the structural transmembrane protein claudin-18. Consistent with findings in other organs, loss of JAM-A decreased β1 integrin expression and impaired filamentous actin formation. Using a model of mild systemic endoxotemia induced by i.p. injection of lipopolysaccharide, we report that JAM-A(-/-) mice showed increased susceptibility to pulmonary edema. On injury, the enhanced susceptibility of JAM-A(-/-) mice to edema correlated with increased, transient disruption of claudin-18, zonula occludens 1, and zonula occludens 2 localization to lung tight junctions in situ along with a delay in up-regulation of claudin-4. In contrast, wild-type mice showed no change in lung tight junction morphologic features in response to mild systemic endotoxemia. These findings support a key role of JAM-A in promoting tight junction homeostasis and lung barrier function by coordinating interactions among claudins, the tight junction scaffold, and the cytoskeleton.

Biochemical Analysis of Claudin-Binding Compatibility

Tissue barrier function is directly mediated by tight junction transmembrane proteins known as claudins. Cells that form tight junctions typically express multiple claudin isoforms, which suggests that heterotypic (head-to-head) binding between different claudin isoforms may play a role in regulating paracellular permeability. To test whether claudins are heterotypically compatible, we developed an assay system using HeLa cells, a claudin-null cell line which expresses other tight junction proteins, including occludin, junction adhesion molecule A, and zonula occludens-1, -2, and -3. HeLa cells stably transfected to express different claudins are cocultured, then subsequently analyzed for the ability to coimmunopurify. Using this approach, we have found that claudin-1, claudin-3, and claudin-5 are heterotypically compatible. In contrast, two closely related claudins, claudin-3 and claudin-4, are incompatible. Differential claudin-binding specificity is likely to have downstream effects on the regulation of tight junction composition and permeability.

Abstract 3624: Activation of tumor specific antibody response following systemic delivery of receptor targeted nanoparticles into Balb/c mice bearing mouse mammary tumors

Targeted nanoparticles (NP) offer a great opportunity for developing new cancer imaging agents and targeted drug delivery carriers. Although increasing evidence supports the potential of the targeted NP for early cancer detection and drug delivery, the impact of systemic delivery of targeted NP on the immune response in the tumor has yet to be addressed. The possible immune enhancement effect of NP has attracted great attention to apply various NP for vaccine development since NP can be nonspecifically taken up by macrophages. In this study, we developed epidermal growth factor receptor (EGFR) receptor-targeted dual imaging modality magnetic iron oxide NP (IONP) using single chain antibody to EGFR (ScFvEGFR) without or with chemotherapy drug doxorubicin (Dox) for targeted tumor imaging and therapy of triple negative breast cancer. The NP were injected into normal Balb/c mice or mice bearing 4T1 mammary tumors once per week for three weeks, then serum and tumors were collected. Systemic delivery of the receptor-targeted NP led to accumulation of NP in the tumors and inhibited tumor growth. Using ELISA on tumor cell lysates and intact tumor cells, we found that mouse serum from targeted IONP had the highest level of mammary tumor specific antibody titers compared to mice that received Dox only, non-targeted NP and no treatment control. However, the antibody titer was decreased in the mice that received ScFvEGFR-IONP-Dox. To understand the antibody activation mechanism by targeted delivery of ScFvEGFR-IONP but not ScFvEGFR-IONP-Dox, we examined changes in the amount, phenotype, and localization of antigen presenting macrophages using a pan-macrophage (CD68) and M2 macrophage (CD163) and also dendritic cells (CD83) markers in frozen tumor tissue sections. Results of double labeling immunofluorescence against CD68 and CD163 showed the majority macrophage subtype at the tumor edge was M2 for the tumors treated with ScFvEGFR-IONP with and without Dox. However, in the central tumor area M1 macrophages was the main phenotype in ScFvEGFR -IONP-Dox treated tumors while ScFvEGFR -IONP still expressed majority M2. Also, a higher level of CD83+ cells infiltrated into the central tumor area of ScFvEGFR-IONP compared to ScFvEGFR-IONP-Dox. Results of our study suggest that targeted delivery of ScFvEGFR-IONP into tumor is able to activate intratumoral macrophages and dendritic cells to antigen presentation and stimulate tumor specific antibody responses while ScFvEGFR-IONP-Dox had decreased amounts of CD163 and CD83. Therefore, targeted delivery of NP into tumors and infiltration of antigen-presenting macrophages into the center of tumors are important for the activation of tumor specific antibody responses. This study could have clinical implications in development of NP imaging and therapy agents for cancer detection and treatment. Citation Format: Christina Ward, Weiping Qian, Emmy Yang, Erica Bozeman, Y. Andrew Wang, Lily Yang. Activation of tumor specific antibody response following systemic delivery of receptor targeted nanoparticles into Balb/c mice bearing mouse mammary tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3624. doi:10.1158/1538-7445.AM2014-3624