Tumor Suppressor Scribble Regulates Assembly of Tight Junctions in the Intestinal Epithelium

Formation of the epithelial barrier and apico-basal cell polarity represent two characteristics and mutually dependent features of differentiated epithelial monolayers. They are controlled by special adhesive structures, tight junctions (TJs), and polarity protein complexes that define the apical and the basolateral plasma membrane. The functional interplay between TJs and polarity complexes remains poorly understood. We investigated the role of Scribble, a basolateral polarity protein and known tumor suppressor, in regulating TJs in human intestinal epithelium. Scribble was enriched at TJs in T84 and SK-CO15 intestinal epithelial cell monolayers and sections of normal human colonic mucosa. siRNA-mediated knockdown of Scribble in SK-CO15 cells attenuated development of epithelial barrier and inhibited TJ reassembly independently of other basolateral polarity proteins Lgl-1 and Dlg-1. Scribble selectively co-imunoprecipitated with TJ protein ZO-1, and ZO-1 was important for Scribble recruitment to intercellular junctions and TJ reassembly. Lastly, Scribble was mislocalized from TJs and its expression down-regulated in interferon-γ-treated T84 cell monolayers and inflamed human intestinal mucosa in vivo. We conclude that Scribble is an important regulator of TJ functions and plasticity in the intestinal epithelium. Down-regulation of Scribble may mediate mucosal barrier breakdown during intestinal inflammation.The epithelial cell layer in the gut plays two crucial physiological functions. One function involves the formation of the physical barrier that separates body compartments from the gut lumen and protects underlying tissues from pathogen invasion and other harmful external stimuli.^1,2 Another function involves the regulation of bidirectional passages of solutes and macromolecules, which is essential for nutrients supply and removal of body waste.^3–5 Both barrier integrity and vectorial transport in the intestinal epithelium are regulated by specialized cellular structures known as tight junctions (TJs). TJs represent a complex network of protein fibrils within the plasma membrane, which encircle the apical region of the epithelial cell perimeter in close proximity to the gut lumen.⁶ TJ fibrils are composed of adhesive transmembrane proteins, which associate with ensembles of scaffolding proteins at the cytosolic face of the membrane. The paracellular barrier is created by homotypical interactions between transmembrane TJ components of contacting epithelial cells such as occludin, members of the claudin family and junctional adhesion molecule-A (JAM-A).^6–8 These cell-cell adhesions are enhanced and regulated by cytosolic scaffolds such as members of zonula occludens (ZO) family and AF-6/afadin, which cluster and stabilize transmembrane TJ components at the plasma membrane.^6–8 Although other junctional complexes at the plasma membrane, viz., adherens junctions (AJs) and desmosomes also mediate cell-cell adhesions, TJs play a unique role in sealing the paracellular space and creating the epithelial barrier.^1,6,7The mature TJs not only mediate barrier function of the intestinal epithelium, but also contribute to the formation and maintenance of the apico-basal cell polarity.^9–11 Such cell polarity implies that the apical and basolateral domains of the plasma membrane differ in the composition of transporters, channels and receptors; therefore ensuring directionality of secretion and adsorption processes in epithelial cells.^12,13 TJs regulate the epithelial cell polarity by creating a fence that prevents intermixing of protein and lipid constituents of the apical and basolateral plasma membrane domains.^9–11 However, TJs alone are not sufficient for the apico-basal polarization of epithelial cells. In this process apical junctions cooperate with specialized protein polarity complexes that control the “identity” of distinct plasma membrane domains.Epithelial cells have three major evolutionarily conserved polarity complexes that were initially identified and named in model invertebrates. They are known as Crumbs (composed by Crumbs, PALS, and PATJ proteins), Par (Par3, Par6, and atypical protein kinase C) and Scribble (including Scribble, Disks Large (Dlg) and Lethal Giant Larvae (Lgl)) complexes.^11,14–16 Crumbs and Par cooperate to define the apical plasma membrane, whereas Scribble is critical for establishment of the basolateral membrane domain.^11,14–16 A number of studies have demonstrated a functional interplay between epithelial junctions and the polarity complexes, where these entities mutually affect each other. Thus, several members of the Crumbs and Par complexes were shown to regulate TJ assembly via either direct binding to TJ components (ZO-1 and claudins) or indirect mechanisms, involving modulation of vesicle trafficking and actin cytoskeleton remodeling.^11,14–16 In contrast, the role of the Scribble polarity complex in the regulation of epithelial TJs is not well understood,^17,18 although such a role is supported by several lines of evidence. For example, mutations in any member of this complex in Drosophila resulted in dramatic disorganization of epithelial architecture that included loss of columnar cell shape and cell-cell adhesions.^19–21 Furthermore, several reports have linked decreased protein levels of mammalian Scribble and Lgl with progression and invasiveness of epithelial tumors,^22–24 which is also accompanied by down-regulation of TJs.²⁵ Two recent studies have addressed the role of Scribble in the regulation cell-cell adhesions in mammalian epithelia; however their results appear to be inconsistent. Indeed, siRNA-mediated depletion of this protein in Madin-Darby canine kidney (MDCK) epithelial cells resulted in altered cell morphology and disorganized E-cadherin-based AJs.²⁶ However, no changes in cell morphology or AJ structure were observed following the silencing of Scribble expression in MCF10A human mammary epithelial cells.²⁷ Such inconsistent results may reflect tissue- specific effects of Scribble depletion, and they indicate that more work is needed to establish functional links between Scribble and TJs in human epithelia under normal physiological conditions and in disease states.In this study, we examined the role of Scribble in the regulation of the intestinal epithelial barrier and reorganization of TJs. Our results demonstrate that Scribble is important for TJ barrier function and assembly, and that it may regulate junctions by interacting with the TJ scaffold, ZO-1. We also report that Scribble is mislocalized and its expression down-regulated in the intestinal epithelium by inflammatory conditions in vitro and in vivo.     

The ErbB4 Ligand Neuregulin-4 Protects against Experimental Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) affects up to 10% of premature infants, has a mortality of 30%, and can leave surviving patients with significant morbidity. Neuregulin-4 (NRG4) is an ErbB4-specific ligand that promotes epithelial cell survival. Thus, this pathway could be protective in diseases such as NEC, in which epithelial cell death is a major pathologic feature. We sought to determine whether NRG4-ErbB4 signaling is protective in experimental NEC. NRG4 was used i) in the newborn rat formula feeding/hypoxia model; ii) in a recently developed model in which 14- to 16-day-old mice are injected with dithizone to induce Paneth cell loss, followed by Klebsiella pneumoniae infection to induce intestinal injury; and iii) in bacterially infected IEC-6 cells in vitro. NRG4 reduced NEC incidence and severity in the formula feed/hypoxia rat model. It also reduced Paneth cell ablation–induced NEC and prevented dithizone-induced Paneth cell loss in mice. In vitro, cultured ErbB4^−/− ileal epithelial enteroids had reduced Paneth cell markers and were highly sensitive to inflammatory cytokines. Furthermore, NRG4 blocked, through a Src-dependent pathway, Cronobacter muytjensii–induced IEC-6 cell apoptosis. The potential clinical relevance of these findings was demonstrated by the observation that NRG4 and its receptor ErbB4 are present in human breast milk and developing human intestine, respectively. Thus, NRG4-ErbB4 signaling may be a novel pathway for therapeutic intervention or prevention in NEC.Necrotizing enterocolitis (NEC) is a devastating intestinal disease primarily affecting premature infants. In the United States, NEC afflicts 7% of infants weighing <1500 g.¹ In addition to prematurity, risk factors include hypoxia, bacterial colonization of the intestine, and formula feeding.² The development of NEC seems to be multifactorial, and patients may have any combination of risk factors at the time of presentation. The current disease model is that the immature gut barrier, along with defects in endogenous antimicrobial activity,³ allows bacterial translocation across the epithelium, triggering an inflammatory response that further worsens gut barrier function. Pathogenic bacteria,^{4, 5} inflammatory cytokines such as tumor necrosis factor (TNF),^{6, 7, 8} and Paneth cell dropout³ have all been associated with human NEC and contribute to NEC-like injury in animal models.Available therapy for either prevention or treatment of NEC is limited, and patients currently face a mortality rate of approximately 30%.^{9, 10, 11} Breast-fed infants have a lower risk of NEC than their formula-fed peers,^{12, 13} and a variety of studies have attempted to identify and characterize factors in human milk that confer this protection. Candidate protective molecules to date include immunoglobulins, oligosaccharides, lactoferrin, and soluble growth factors, such as epidermal growth factor (EGF)¹⁴ and heparin-binding EGF-like growth factor (HB-EGF).¹⁵ In rat and mouse models, enteral administration of either EGF^{16, 17} or HB-EGF¹⁸ decreases the incidence and severity of NEC. The primary receptor for both EGF and HB-EGF is EGF receptor (EGFR), the prototypic member of the ErbB receptor tyrosine kinase family. However, HB-EGF also activates ErbB4, a member of the ErbB family whose potential role in the developing gut and NEC is not known.ErbB4 has unique biochemical properties distinguishing it from other ErbB family members. Compared with EGFR, ErbB2, or ErbB3, it recognizes a broader collection of ligands, including the EGF-like growth factors HB-EGF and betacellulin as well as the heregulin/neuregulin molecules.¹⁹ At the same time, the ErbB4 c-terminus contains a distinct and somewhat restricted set of functional docking sites for downstream effectors²⁰ and is thus predicted to elicit divergent cellular effects on activation versus other family members. In fact, we recently demonstrated that neuregulin-4 (NRG4), an ErbB4-specific ligand that does not bind or activate other family members, including EGFR,²¹ specifically promotes survival but not migration or proliferation of mouse colon epithelial cells.²² Thus, ErbB4 is a potentially unique and selective target for therapeutic protection in diseases in which intestinal epithelial cell death is a major pathologic feature.We previously reported that ErbB4 is up-regulated in adult human and murine colon inflammation in vivo²³ and that ErbB4 overexpression protects cultured colonocytes from cytokine-induced apoptosis in a ligand-dependent manner.²⁴ Furthermore, i.p. NRG4 administration reduces the severity of acute murine dextran sulfate sodium colitis.²² Thus, it seems that ErbB4 induction is a natural compensatory response meant to preserve the epithelium rather than part of disease pathology and that ErbB4 activation with exogenous ligand is protective against induced inflammation. However, the role of this signaling pathway in the small intestine, or during development, has not been described. We hypothesized that ErbB4 and its ligands have a protective role in the small bowel during postnatal development, particularly in the setting of NEC-associated acute injury and inflammation. To advance our understanding of ErbB4 biology in intestinal homeostasis and disease, we tested the hypothesis that NRG4-ErbB4 signaling is protective in experimental NEC.     

Mutation in Osteoactivin Decreases Bone Formation in Vivo and Osteoblast Differentiation in Vitro

We have previously identified osteoactivin (OA), encoded by Gpnmb, as an osteogenic factor that stimulates osteoblast differentiation in vitro. To elucidate the importance of OA in osteogenesis, we characterized the skeletal phenotype of a mouse model, DBA/2J (D2J) with a loss-of-function mutation in Gpnmb. Microtomography of D2J mice showed decreased trabecular mass, compared to that in wild-type mice [DBA/2J-Gpnmb⁺/SjJ (D2J/Gpnmb⁺)]. Serum analysis showed decreases in OA and the bone-formation markers alkaline phosphatase and osteocalcin in D2J mice. Although D2J mice showed decreased osteoid and mineralization surfaces, their osteoblasts were increased in number, compared to D2J/Gpnmb⁺ mice. We then examined the ability of D2J osteoblasts to differentiate in culture, where their differentiation and function were decreased, as evidenced by low alkaline phosphatase activity and matrix mineralization. Quantitative RT-PCR analyses confirmed the decreased expression of differentiation markers in D2J osteoblasts. In vitro, D2J osteoblasts proliferated and survived significantly less, compared to D2J/Gpnmb⁺ osteoblasts. Next, we investigated whether mutant OA protein induces endoplasmic reticulum stress in D2J osteoblasts. Neither endoplasmic reticulum stress markers nor endoplasmic reticulum ultrastructure were altered in D2J osteoblasts. Finally, we assessed underlying mechanisms that might alter proliferation of D2J osteoblasts. Interestingly, TGF-β receptors and Smad-2/3 phosphorylation were up-regulated in D2J osteoblasts, suggesting that OA contributes to TGF-β signaling. These data confirm the anabolic role of OA in postnatal bone formation.Osteoporosis is a growing public health problem, in part because of the increasing numbers of people living beyond the age of 65 years.¹ It is characterized by low bone mass due to increased bone resorption by osteoclasts and decreased bone formation by osteoblasts, with significant deterioration in the bone microarchitecture leading to high bone fragility and increased fracture risk.^1,2 The net effect of osteoporosis is low bone mass.¹ There is an increasing demand for identifying novel bone anabolic factors with potential therapeutic benefits in treating generalized bone loss, such as osteoporosis and/or major skeletal fracture.Osteoactivin is a novel glycoprotein first identified in natural mutant osteopetrotic rats.³ The same protein has been identified and named separately in several other species: as dendritic cell heparan sulfate proteoglycan integrin dependent ligand (DCHIL) in mouse dendritic cells,⁴ as transmembrane glycoprotein NMB (GPNMB) in human melanoma cell lines and melanocytes,⁵ and as hematopoietic growth factor inducible neurokinin (HGFIN) in human tumor cells.⁶ The current recommended name for the protein encoded by Gpnmb in mouse is transmembrane glycoprotein NMB (http://www.ncbi.nlm.nih.gov/protein/Q99P91.2); here, we continue to use osteoactivin (OA) for the protein and Gpnmb for the gene. OA is a type I transmembrane protein that consists of multiple domains, including an extracellular domain, transmembrane domain, and protein sorting signal sequence.⁷ Within the C-terminal domain, OA has an RGD motif, predicting an integrin attachment site.^3,7–9Our research group initially reported on the novel role of OA in osteoblast differentiation and function.^7–10 We demonstrated that OA expression has a temporal pattern during osteoblast differentiation, being highest during matrix maturation and culture mineralization in vitro.^7–11 Using loss-of–function and gain-of–function approaches in osteoblasts, we reported that OA overexpression increases osteoblast differentiation and function and that OA down-regulation decreases nodule formation, alkaline phosphatase (ALP) activity, osteocalcin (OC) production, and matrix mineralization in vitro.⁷ We also reported on the positive role of OA in mesenchymal stem cell (MSCs) differentiation into osteoblasts in vitro.¹² In another study, we showed that recombinant OA protein induces higher osteogenic potential of fetal-derived MSCs, compared with bone marrow–derived MSCs¹³ and its osteogenic effects in the mouse C3H10T1/2 MSC cell line were similar to those of recombinant BMP-2.¹² We also localized OA protein as associated predominately with osteoblasts lining trabecular bones in vivo,¹¹ and showed that local injection of recombinant OA increased bone mass in a rat model.¹⁴ Moreover, in a fracture repair model OA expression increased over time, reaching a maximum 2 weeks after fracture.¹¹ In a parallel study, recombinant OA supported bone regeneration and formation in a rat critical-size calvarial defect model.¹⁵ Others have shown that OA is highly expressed by osteoclasts in vitro, suggesting that it may regulate osteoclast formation and activity.¹⁶There is urgent need for an animal model to fully examine the role of OA in osteogenesis. Interestingly, a natural mutation of the Gpnmb gene has been identified in the DBA/2J (D2J) mouse strain.¹⁷ These mice exhibit high-frequency hearing loss, which begins at the time of weaning and becomes severe by 2 to 3 months of age.^18,19 Aged D2J mice also develop progressive eye abnormalities that closely mimic human hereditary glaucoma. The onset of disease symptoms falls roughly between 3 and 4 months of age, and disease becomes severe by 6 months of age.^5,20 D2J mice are homozygous for a nonsense mutation in the Gpnmb gene sequence that induces an early stop codon, generating a truncated protein sequence of 150 amino acids (aa) instead of the full-length 562-aa OA protein.⁵ The control for the D2J mouse is the wild-type DBA/2J-Gpnmb⁺/SjJ mouse (D2J/Gpnmb⁺), homozygous for the wild-type Gpnmb gene.²¹ These Gpnmb wild-type mice do not develop glaucoma, as D2J mice do, although they exhibit mild iris stromal atrophy.²¹In the present study, we used Gpnmb mutant (D2J) and Gpnmb wild-type (D2J/Gpnmb⁺) mice to gain insight into the role of OA in osteogenesis and in osteoblast differentiation and function. Here, we report that loss-of–function mutation of Gpnmb suppresses bone formation by directly affecting osteoblast proliferation and survival, leading to a decreased number of differentiated osteoblasts with suppressed activity in bone mineralization. Thus, our data point to OA as a novel and positive regulator of postnatal bone formation.     

Inhibition of AMP-Activated Protein Kinase Accentuates Lipopolysaccharide-Induced Lung Endothelial Barrier Dysfunction and Lung Injury in Vivo

The aim of this study was to determine the role of AMP-activated protein kinase (AMPK) in lipopolysaccharide (LPS)-induced lung endothelial barrier dysfunction and lung injury in vivo. Both cultured human pulmonary artery endothelial cells (HPAECs) and experimental animals [AMPK subunit α–deficient mice and wild-type (WT) control mice (C57BL/6J)] were used. In cultured HPAECs, LPS increased endothelial permeability in parallel with a decrease in AMPK activity. Consistent with this observation, AMPK activation with the potent AMPK activator 5-aminoimidazole-4-carboxamide-1-d-ribofuranoside (AICAR) attenuated LPS-induced endothelial hyperpermeability in vitro. Intratracheal administration of LPS (1 mg/kg) in WT mice reduced AMPK phosphorylation at Thr172 in lung tissue extracts, increased protein content and cell count in bronchial alveolar lavage fluid, and increased Evans Blue dye infiltration into the lung. These same attributes were similarly enhanced in AMPKα-knockout mice, compared with WT mice. Pretreatment with AICAR reduced these lung injury indicators in LPS-treated WT mice. AMPK activation with AICAR attenuated LPS-induced endothelial hyperpermeability by activating the Rac/Cdc42/PAK pathway, with concomitant inhibition of the Rho pathway, and decreased VE-cadherin phosphorylation at Tyr658. We conclude that AMPK activity supports normal endothelial barrier function and that LPS exposure inhibits AMPK, thereby contributing to endothelial barrier dysfunction and lung injury.Vascular endothelial permeability plays a pivotal role in regulating many physiological and pathological processes, including angiogenesis, immunity, and inflammation.¹ In lungs, endothelial cells form a semipermeable barrier between the vessel lumen and underlying alveoli, thereby mediating the transmigration of blood cells and maintaining fluid homeostasis. The integrity of the endothelial cell (EC) monolayer therefore directly determines lung vascular permeability. For example, EC barrier dysfunction can lead to an increase in permeation of fluid and macromolecules into the interstitium and alveolar space, resulting in pulmonary edema, a major characteristic of acute lung injury.RhoA, Rac1, and Cdc42 are key members of the Rho GTPase family and are activated on binding GTP at the membrane.² These proteins are intimately involved in regulating cell adhesion and cytoskeletal dynamics, both of which play an important role in endothelial barrier function.^3–5 For example, Rac1 and Cdc42 are important in maintaining, stabilizing, and restoring the endothelial barrier.³ More specifically, Baumer et al⁶ demonstrated that Rac1 is involved in mitigating endothelial hyperpermeability by a subset of agonists, including thrombin and lipopolysaccharide (LPS). In addition, activation of the Rho GTPases Cdc42 and Rac1 restores endothelium integrity after lung injury.⁷ Although the contributions of Rho GTPases in maintaining endothelial barrier function are well established, how Rho GTPase is regulated in endothelial cells is largely unknown.AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine kinase; a catalytic α subunit and regulatory β and γ subunits are important in maintaining the stability of the complex. AMPK belongs to a family of energy-sensing enzymes that function as fuel gauges, monitoring changes in the energy status of the cell.⁸ AMPK is activated in response to a variety of stressors that increase the intracellular ratio of AMP to ATP. On activation, AMPK phosphorylates a number of downstream targets, thereby affecting glucose metabolism, fatty acid oxidation, hepatic lipogenesis, and cholesterol synthesis.⁹ In addition to its regulatory role in metabolism, recent studies have demonstrated a role for AMPK in maintaining normal endothelial function.¹⁰ For example, AMPK subunit α2 exerts protective effects against atherosclerosis by inhibiting the endoplasmic reticulum stress response in ECs.¹¹ Thus, agents that enhance EC barrier function are of potential therapeutic value in a variety of pathological settings, including inflammatory disease, atherosclerosis, and tumor angiogenesis. The effects of AMPK on endothelial barrier function and vascular permeability have not been investigated previously. Thus, the aim of the present study was to investigate whether AMPK protects lung endothelial barrier function and mitigates acute lung injury in response to LPS.     

Lactobacillus acidophilus Induces a Strain-specific and Toll-Like Receptor 2–Dependent Enhancement of Intestinal Epithelial Tight Junction Barrier and Protection Against Intestinal Inflammation

Defective intestinal tight junction (TJ) barrier is an important pathogenic factor of inflammatory bowel disease. To date, no effective therapies that specifically target the intestinal TJ barrier are available. The purpose of this study was to identify probiotic bacterial species or strains that induce a rapid and sustained enhancement of intestinal TJ barrier and protect against the development of intestinal inflammation by targeting the TJ barrier. After high-throughput screening of >20 Lactobacillus and other probiotic bacterial species or strains, a specific strain of Lactobacillus acidophilus, referred to as LA1, uniquely produced a marked enhancement of the intestinal TJ barrier. LA1 attached to the apical membrane surface of intestinal epithelial cells in a Toll-like receptor (TLR)-2–dependent manner and caused a rapid increase in enterocyte TLR-2 membrane expression and TLR-2/TLR-1 and TLR-2/TLR-6 hetero-complex–dependent enhancement in intestinal TJ barrier function. Oral administration of LA1 caused a rapid enhancement in mouse intestinal TJ barrier, protected against a dextran sodium sulfate (DSS) increase in intestinal permeability, and prevented the DSS-induced colitis in a TLR-2– and intestinal TJ barrier–dependent manner. In conclusion, we report for the first time that a specific strain of LA causes a strain-specific enhancement of intestinal TJ barrier through a novel mechanism that involves the TLR-2 receptor complex and protects against the DSS-induced colitis by targeting the intestinal TJ barrier.

Intestinal epithelial tight junctions (TJs) are the apical-most junctional complexes and act as a functional and structural barrier against the paracellular permeation of harmful luminal antigens, which promote intestinal inflammation.¹ The increased intestinal permeability caused by defective intestinal epithelial TJ barrier or a leaky gut is an important pathogenic factor that contributes to the development of intestinal inflammation in inflammatory bowel disease (IBD) and other inflammatory conditions of the gut, including necrotizing enterocolitis and celiac disease.^2,3 Clinical studies in patients with IBD have found that a persistent increase in intestinal permeability after clinical remission is predictive of poor clinical outcome and early recurrence of the disease, whereas normalization of intestinal permeability correlates with a sustained long-term clinical remission.4, 5, 6 Accumulating evidence has found that a defective intestinal TJ barrier plays an important role in exacerbation and prolongation of intestinal inflammation in IBD. Currently, no effective therapies that specifically target the tightening of the intestinal TJ barrier are available.Intestinal microbiota play an important role in modulating the immune system and in the pathogenesis of intestinal inflammation.⁷ Patients with IBD have bacterial dysbiosis in the gut, characterized by a decrease in bacterial diversity and an aberrant increase in some commensal bacteria, which are an important factor in the pathogenesis of intestinal inflammation.^8,9 Normal microbial flora of the gastrointestinal tract consists both of bacteria that are known to have beneficial effects (probiotic bacteria) on intestinal homeostasis and bacteria that could potentially have detrimental effects on gut health (pathogenic bacteria).¹⁰ The modulation of intestinal microflora affects the physiologic and pathologic states in humans and animals. For example, fecal transplantation from healthy, unaffected individuals to patients with refractory Clostridium difficile colitis is curative in up to 94% of the treated patients, and transfer of stool microbiome from obese mice induces obesity in previous lean mice, whereas transfer of microbiome from lean mice preserves the lean phenotype.11, 12, 13 The beneficial effects of gut microbiota are host and bacterial species-specific.¹⁴ Although multiple studies indicate that some commensal bacteria play a beneficial role in gut homeostasis by preserving or promoting the intestinal barrier function, because of conflicting reports, it remains unclear which probiotic species cause a persistent predictable enhancement in the TJ barrier and could be used to treat intestinal inflammation by targeting the TJ barrier. For example, some studies suggest that Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus plantarum, or Lactobacillus rhamnosus cause a modest enhancement in the intestinal epithelial TJ barrier, whereas others have found minimal or no effect of these probiotic species on the intestinal TJ barrier.15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 The major aim the current study was to perform a high-throughput screening of Lactobacillus and other bacterial species to identify probiotic species that induce a rapid, predictable, and marked increase in the intestinal epithelial TJ barrier and protect against the development of intestinal inflammation by preserving the intestinal TJ barrier.In the studies described herein, most of the probiotic species tested (>20 species or strains) had a modest or minimal effect on intestinal TJ barrier function. L. acidophilus uniquely caused a rapid and marked increase in intestinal TJ barrier function. Further analysis indicated that the effect of L. acidophilus was strain-specific, limited to a specific strain of L. acidophilus, and did not extend to other L. acidophilus strains. The L. acidophilus enhancement of the intestinal TJ barrier was mediated by live bacterial-enterocyte interaction that involved Toll-like receptor (TLR)-2 heterodimeric complexes on the apical membrane surface of intestinal epithelial cells. Our animal studies also found that L. acidophilus causes a marked enhancement in mouse intestinal barrier function and protects against the dextran sodium sulfate (DSS)–induced colitis by preserving and augmenting the mouse intestinal barrier function in a strain-specific manner.     

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.To support efficient gas exchange, the lung must maintain a barrier between the atmosphere and fluid-filled tissues. Without this crucial barrier, the air spaces would flood, and gas exchange would be severely limited.^{1, 2} In acute lung injury and acute respiratory distress syndrome, fluid leakage into the lung air space is associated with increased patient mortality and morbidity.^{3, 4} Lung fluid clearance is maintained, in part, by tight junctions that regulate paracellular flux between cells.^{5, 6, 7}Tight junctions are multiprotein complexes located at sites of cell-cell contact and are composed of transmembrane, cytosolic, and cytoskeletal proteins that together produce a selective barrier to water, ions, and soluble molecules. Among the transmembrane proteins required for epithelial barrier function is the Ig superfamily protein junctional adhesion molecule A (JAM-A).^{8, 9, 10, 11} JAM-A is ubiquitously expressed and regulates several processes related to cell-cell and cell-matrix interactions, including cell migration and proliferation in addition to barrier function regulation. Specific mechanistic roles for JAM-A in regulating tight junctions continue to be elucidated.JAM-A signaling is stimulated by cis-dimerization, which provides a platform for multiple proteins to cluster in close apposition.¹² In particular, JAM-A has been shown to recruit scaffold proteins, such as zonula occludens 1 (ZO-1), ZO-2, and Par3, to tight junctions, where these proteins enhance the assembly of multiprotein junctional complexes.^{13, 14} More recently, it was demonstrated that JAM-A directly interacts with ZO-2, which then recruits other scaffold proteins, including ZO-1.¹⁵ This nucleates a core complex that includes afadin, PDZ-GEF1, and Rap2c and that stabilizes filamentous actin by repressing rhoA.¹⁵ Together, all of these activities of JAM-A promote tight junction formation and barrier function.Although JAM-A is part of the tight junction complex, the main structural determinants of the paracellular barrier are proteins known as claudins. Claudins are a family of transmembrane proteins that interact to form paracellular channels that either promote or limit paracellular ion and water flux.^{16, 17, 18} Claudins that promote flux are known collectively as pore-forming claudins, whereas claudins that limit flux are known as sealing claudins.¹⁹ In fact, there is a link between JAM-A and claudin expression because it was demonstrated that JAM-A–deficient intestinal epithelium has increased expression of two pore-forming claudins, claudin-10 and claudin-15.²⁰ Critically, increased claudin-10 and claudin-15 leads to a compromised intestinal barrier, as demonstrated by an enhanced susceptibility of JAM-A^−/− mice to dextran sulfate sodium–induced colitis.²⁰ However, it is not known whether this relationship between JAM-A and claudin expression occurs in other classes of epithelia.Several claudins are expressed by the alveolar epithelium. The most prominent alveolar claudins are claudin-3, claudin-4, and claudin-18; several additional claudins are expressed by alveolar epithelium and throughout the lung as well.^{21, 22} A central role for claudin-18 in regulating lung barrier function was demonstrated in two independently derived strains of claudin-18–deficient mice that showed altered alveolar tight junction morphologic features and increased paracellular permeability.^{23, 24} Claudin-4 also is an important part of the lung response to acute lung injury because it improves barrier function by limiting alveolar epithelial permeability and promoting lung fluid clearance.^{25, 26} Although claudin-4–deficient mice show a relatively mild baseline phenotype, these mice have impaired fluid clearance in response to ventilator-induced lung injury.²⁷ An analysis of ex vivo perfused human donor lungs revealed that increased claudin-4 was linked to increased rates of alveolar fluid clearance and decreased physiologic respiratory impairment,²⁸ further underscoring the importance of claudin regulation in promoting efficient barrier function in response to injury.Although JAM-A has a clear role in regulating gut permeability,²⁰ a recent report that wild-type and JAM-A^−/− mice show comparable levels of pulmonary edema in response to intratracheal endotoxin challenge²⁹ raises questions about potential roles for JAM-A in lung barrier function. Herein we used a combination of in vivo and in vitro approaches to assess the contributions of JAM-A to alveolar barrier function. Using a model of mild systemic endotoxemia induced by i.p. injection of Escherichia coli–derived lipopolysaccharide (LPS), we found that JAM-A^−/− mice showed greater lung edema than comparably treated wild-type mice. Greater sensitivity to injury was due to aberrant regulation of tight junction protein expression, which was recapitulated by JAM-A–depleted alveolar epithelial cells. JAM-A depletion also resulted in decreased β1 integrin protein levels and disrupted cytoskeletal assembly. Together, these effects indicated that the loss of JAM-A impaired tight junction formation, thus rendering the lung more susceptible to edema and injury.     

Hypercholesterolemia Induces Angiogenesis and Accelerates Growth of Breast Tumors in Vivo

Obesity and metabolic syndrome are linked to an increased prevalence of breast cancer among postmenopausal women. A common feature of obesity, metabolic syndrome, and a Western diet rich in saturated fat is a high level of circulating cholesterol. Epidemiological reports investigating the relationship between high circulating cholesterol levels, cholesterol-lowering drugs, and breast cancer are conflicting. Here, we modeled this complex condition in a well-controlled, preclinical animal model using innovative isocaloric diets. Female severe combined immunodeficient mice were fed a low-fat/no-cholesterol diet and then randomized to four isocaloric diet groups: low-fat/no-cholesterol diet, with or without ezetimibe (cholesterol-lowering drug), and high-fat/high-cholesterol diet, with or without ezetimibe. Mice were implanted orthotopically with MDA-MB-231 cells. Breast tumors from animals fed the high-fat/high-cholesterol diet exhibited the fastest progression. Significant differences in serum cholesterol level between groups were achieved and maintained throughout the study; however, no differences were observed in intratumoral cholesterol levels. To determine the mechanism of cholesterol-induced tumor progression, we analyzed tumor proliferation, apoptosis, and angiogenesis and found a significantly greater percentage of proliferating cells from mice fed the high-fat/high-cholesterol diet. Tumors from hypercholesterolemic animals displayed significantly less apoptosis compared with the other groups. Tumors from high-fat/high-cholesterol mice had significantly higher microvessel density compared with tumors from the other groups. These results demonstrate that hypercholesterolemia induces angiogenesis and accelerates breast tumor growth in vivo.Breast cancer is the most frequent and second most deadly cancer in women, with >230,000 new cases and nearly 40,000 estimated annual deaths in the United States alone.¹ Most breast cancers are invasive ductal carcinomas that have penetrated through the ductal wall and have invaded surrounding breast tissue, often leading to metastasis to other sites, including the lung, liver, and bone, resulting in significant morbidity, suffering, and death.Breast cancer rates differ significantly among nations worldwide, with developed countries in North America and Northern and Western Europe reporting the highest incidence of breast cancer globally. The incidence of breast cancer in these regions is three to four times higher than the incidence of breast cancer in parts of Asia and Africa.¹ More important, relocation studies have shown that women who move from an area of low incidence of breast cancer (Japan) to a high-incidence area (Los Angeles, CA) develop breast cancer at rates similar to that of U.S. women, with the rate of breast cancer incidence associated with the time of life at immigration.² For Japanese women who immigrated early in life, the rate of breast cancer was significantly higher than in women who moved from Japan to Los Angeles later in life, with both groups demonstrating a significantly greater incidence of breast cancer compared with women in their homeland.² These findings point to a possible dietary factor in a traditional Western diet that may be contributing to increased breast cancer incidence.² Although specific dietary factors that increase the rate of breast cancer among U.S. women are not well understood, some evidence suggests a significant, positive association between breast cancer incidence and intake of saturated fat in postmenopausal women.³ Moreover, both adult weight gain and metabolic syndrome are linked to an increased prevalence of breast cancer.^4–6 A common feature shared by adult obesity, metabolic syndrome, and a Western diet rich in fat is a high level of circulating cholesterol.Similar to other mammalian cells, breast ductal epithelial cells synthesize cholesterol endogenously via the mevalonate pathway and absorb cholesterol-containing lipoproteins from the circulation. Consequently, control of cellular cholesterol content is achieved by regulating systemic cholesterol concentration and by balancing intrinsic cellular metabolic processes.^7,8 Yet, despite the sophisticated mechanisms for maintaining the appropriate cholesterol balance, it has been reported that ductal epithelium of the breast may be sensitive to systemic changes in serum cholesterol levels.⁹ These changes may result in aberrant cellular responses to specific growth factors and/or cytokines, resulting in altered patterns of cellular growth, apoptosis, morphological characteristics, and intercellular communication.Retrospective epidemiological studies have suggested that elevated serum cholesterol and/or hyperlipidemia increase the rate of breast cancer among women of developed countries.^4,5,10 These case-controlled, cohort, or comparative studies also reported that statin drugs, which inhibit the rate-limiting step in cholesterol synthesis¹¹ or the hydrophobic subclass of statins, decrease overall breast cancer prevalence,^12–15 reduce the rate of developing an estrogen receptor/progesterone receptor–negative cancer, and promote a downward shift in breast cancer grade and stage.¹⁶ However, results from several large, prospective, epidemiological studies examining the relationship between serum cholesterol levels, cholesterol-lowering drugs, and breast cancer incidence have been inconclusive.^14,17,18 For example, it has been shown that women with clinically high (≥240 mg/dL) fasting total serum cholesterol have a slight, but statistically significant, increased incidence of breast cancer.¹⁸ A separate study demonstrated that untreated, self-reported high cholesterol was not associated with an increased rate of breast cancer, even among obese and postmenopausal women.¹⁴ A third study found that the hydrophobic class of statins significantly reduced the incidence of invasive breast cancer among older women.^13,15In contrast to the previously described studies,¹⁷ which suggested an association between statin use and decreased incidence of breast cancer, a prospective study examining the effect of cholesterol-lowering drug use on many cancers showed that the incidence of breast cancer was no different among women reporting long-term statin use compared with women who reported never having used statins. This latter report adjusted for age and history of high cholesterol levels but did not separate women on the basis of menopausal status.Although the effect of statins on the incidence of primary breast cancer remains unclear, epidemiological studies report that lipophilic statin use among women diagnosed with stage I to III breast cancer was associated with a reduced rate of cancer recurrence.^19–21 Taken together, these numerous, inconsistent, and sometimes contradicting epidemiological results highlight an important need for studies that model the complex relationship between serum cholesterol, cholesterol-lowering drugs, and breast cancer using well-controlled, preclinical models.As a function of the suggested links between postmenopausal weight gain, metabolic syndrome, and breast cancer,^4–6 prior work describing an effect of elevated cholesterol and statin drugs on breast cancer risk,^4,5,12–14 and our own reports demonstrating a role for cholesterol in prostate cancer progression,^22–25 we chose to determine whether hypercholesterolemia would promote breast cancer progression in an in vivo model that tightly controlled other metabolic variables associated with a Western diet.It has been previously reported that a high-fat Western diet promoted the growth of breast tumors in the MMTV-PyMT breast cancer model in which tumor formation is driven by polyoma middle T antigen²⁶ and in the apolipoprotein E (ApoE) knockout mouse.²⁷ Although these studies suggested that a Western diet promotes breast cancer growth, a specific role for cholesterol was not established because isocaloric diets were not compared and cholesterol was not specifically targeted (as we have done in the current study).By using novel diet and feeding strategies, we have generated an innovative isocaloric diet approach in which we are able to determine the specific effects of cholesterol in mice. This approach provides the opportunity to study the effects of hypercholesterolemia on tumor growth. In the current study, we apply this approach to an orthotopic xenograft model of human estrogen receptor–negative breast cancer and demonstrate that a hypercholesterolemic diet promotes the growth of breast cancer. This effect is blocked by the hypocholesterolemic drug ezetimibe. Histological and biochemical analyses indicate that hypercholesterolemia increased cell proliferation and tumor cell apoptosis, and significantly increased tumor angiogenesis. To our knowledge, this is the first report of a specific role for cholesterol in promoting breast cancer growth.     

A Role for Vascular Deficiency in Retinal Pathology in a Mouse Model of Ataxia-Telangiectasia

Ataxia-telangiectasia is a multifaceted syndrome caused by null mutations in the ATM gene, which encodes the protein kinase ATM, a key participant in the DNA damage response. Retinal neurons are highly susceptible to DNA damage because they are terminally differentiated and have the highest metabolic activity in the central nervous system. In this study, we characterized the retina in young and aged Atm-deficient mice (Atm^−/−). At 2 months of age, angiography revealed faint retinal vasculature in Atm^−/− animals relative to wild-type controls. This finding was accompanied by increased expression of vascular endothelial growth factor protein and mRNA. Fibrinogen, generally absent from wild-type retinal tissue, was evident in Atm^−/− retinas, whereas mRNA of the tight junction protein occludin was significantly decreased. Immunohistochemistry labeling for occludin in 6-month-old mice showed that this decrease persists in advanced stages of the disease. Concurrently, we noticed vascular leakage in Atm^−/− retinas. Labeling for glial fibrillary acidic protein demonstrated morphological alterations in glial cells in Atm^−/− retinas. Electroretinographic examination revealed amplitude aberrations in 2-month-old Atm^−/− mice, which progressed to significant functional deficits in the older mice. These results suggest that impaired vascularization and astrocyte–endothelial cell interactions in the central nervous system play an important role in the etiology of ataxia-telangiectasia and that vascular abnormalities may underlie or aggravate neurodegeneration.Some brain disorders may have a vascular origin,^1,2 and vascular diseases can be directly linked to neuronal and synaptic dysfunction through changes in the blood flow, increase in blood–brain barrier permeability, and in nutrient supply.³ A healthy brain relies on the proper function and communication of all cells comprising the neurovascular unit: neurons, astrocytes, brain endothelium, and vascular smooth muscle cells.⁴Impaired genomic stability interferes with cellular homeostasis and poses a constant threat to cellular viability.⁵ The cell combats this threat by activating the DNA damage response (DDR), a complex signaling network that detects the DNA lesions, promotes their repair, and temporarily modulates cellular metabolism while the damage is being repaired.⁶ The DDR is vigorously activated by DNA double-strand breaks (DSBs), a particularly cytotoxic DNA lesion induced by ionizing radiation, radiomimetic chemicals, and oxygen radicals.^7,8 The DNA damage response is a hierarchical process executed by sensor/mediator proteins that accumulate at DSB sites and by protein kinases that serve as transducers of the DNA damage alarm to numerous downstream effectors.⁶ The primary transducer of the cellular response to DSBs is the protein kinase ATM, which phosphorylates many key players in the various branches of the DDR.⁹Ataxia-telangiectasia (A-T) is an autosomal recessive disorder caused by mutations in the ATM gene that encodes the ATM protein.¹⁰ A-T is characterized by progressive neurodegeneration affecting mainly the cerebellum, which develops into severe neuromotor dysfunction; peripheral neuropathy; immunodeficiency that spans the B- and T-cell systems; thymic and gonadal atrophy; marked predisposition to lymphoreticular malignancies; and chromosomal fragility and acute sensitivity to ionizing radiation. Cultured ATM-deficient cells exhibit severe cellular sensitivity to DSB-inducing agents, with markedly defective DSB response.DSBs are constantly induced in all body cells by metabolic byproducts such as oxygen radicals. Oxidative stress has been consistently associated with various neurodegenerative conditions.^11–14 Indeed, there is substantial evidence for a role of oxidative damage in the progression of neurodegenerative disorders, including Parkinson''s and Alzheimer''s diseases.¹⁵ Similarly, elevated oxidative stress has been identified in several DNA repair deficiencies, including A-T.^16–22 Notably, ATM has recently been shown to be activated by oxidative stress.²³ Ocular tissues, especially the retina, are exposed to extremely high levels of reactive oxygen species. Nevertheless, retinal neurodegeneration has not been reported in A-T patients. The ocular manifestation of the disease reported to date is scleral telangiectasia^24,25 and saccadic abnormalities.²⁶ Moreover, no retinal pathology has been described to date in Atm-deficient mice despite their increased sensitivity to reactive oxygen species–inducing agents.²⁷ We examined the link between retinal vascular pathology and function in young and aging Atm-deficient mice. We present evidence for vascular changes that accompany neuronal deficiencies in the retina.     

Fatty Acid Ethyl Esters Are Less Toxic Than Their Parent Fatty Acids Generated during Acute Pancreatitis

Although ethanol causes acute pancreatitis (AP) and lipolytic fatty acid (FA) generation worsens AP, the contribution of ethanol metabolites of FAs, ie, FA ethyl esters (FAEEs), to AP outcomes is unclear. Previously, pancreata of dying alcoholics and pancreatic necrosis in severe AP, respectively, showed high FAEEs and FAs, with oleic acid (OA) and its ethyl esters being the most abundant. We thus compared the toxicities of FAEEs and their parent FAs in severe AP. Pancreatic acini and peripheral blood mononuclear cells were exposed to FAs or FAEEs in vitro. The triglyceride of OA (i.e., glyceryl tri-oleate) or OAEE was injected into the pancreatic ducts of rats, and local and systemic severities were studied. Unsaturated FAs at equimolar concentrations to FAEEs induced a larger increase in cytosolic calcium, mitochondrial depolarization, and necro-apoptotic cell death. Glyceryl tri-oleate but not OAEE resulted in 70% mortality with increased serum OA, a severe inflammatory response, worse pancreatic necrosis, and multisystem organ failure. Our data show that FAs are more likely to worsen AP than FAEEs. Our observations correlate well with the high pancreatic FAEE concentrations in alcoholics without pancreatitis and high FA concentrations in pancreatic necrosis. Thus, conversion of FAs to FAEE may ameliorate AP in alcoholics.Although fat necrosis has been associated with severe cases of pancreatitis for more than a century,^{1, 2} and alcohol consumption is a well-known risk factor for acute pancreatitis (AP),³ only recently have we started understanding the mechanistic basis of these observations.^{4, 5, 6, 7} High amounts of unsaturated fatty acids (UFAs) have been noted in the pancreatic necrosis and sera of severe AP (SAP) patients by multiple groups.^{8, 9, 10, 11, 12} These high UFAs seem pathogenically relevant because several studies show UFAs can cause pancreatic acinar injury or can worsen AP.^{11, 12, 13, 14} Ethanol may play a role in AP by distinct mechanisms,³ including a worse inflammatory response to cholecystokinin,⁴ increased zymogen activation,¹⁵ basolateral enzyme release,¹⁶ sensitization to stress,⁷ FA ethyl esters (FAEEs),¹⁷ cytosolic calcium,¹⁸ and cell death.¹⁹Because the nonoxidative ethanol metabolite of fatty acids (FAs), FAEEs, were first noted to be elevated in the pancreata of dying alcoholics, they have been thought to play a role in AP.^{17, 19, 20, 21, 22} Conclusive proof of the role of FAEEs in AP in comparison with their parent UFAs is lacking. Uncontrolled release of lipases into fat, whether in the pancreas or in the peritoneal cavity, may result in fat necrosis, UFA generation, which has been associated with SAP.^{11, 12} Pancreatic homogenates were also noted to have an ability to synthesize FAEEs from FAs and ethanol,^{20, 23} and the putative enzyme for this was thought to be a lipase.^{24, 25} It has been shown that the FAEE synthase activity of the putative enzyme exceeds its lipolytic capacity by several fold.²⁵Triglyceride (TG) forms >80% of the adipocyte mass,^{26, 27, 28} oleic acid (OA) being the most enriched FA.^{9, 29} We recently showed that lipolysis of intrapancreatic TG worsens pancreatitis.^{11, 12} Therefore, after noting the ability of the pancreas to cause lipolysis of TG into FAs and also to have high FAEE synthase activity and FAEE concentrations, we decided to compare the relative ability of FAEEs and their parent FAs to initiate deleterious signaling in pancreatitis and to investigate their impact on the severity of AP.     

Type I Interferon Contributes to Noncanonical Inflammasome Activation,Mediates Immunopathology,and Impairs Protective Immunity during Fatal Infection with Lipopolysaccharide-Negative Ehrlichiae

Ehrlichia species are intracellular bacteria that cause fatal ehrlichiosis, mimicking toxic shock syndrome in humans and mice. Virulent ehrlichiae induce inflammasome activation leading to caspase-1 cleavage and IL-18 secretion, which contribute to development of fatal ehrlichiosis. We show that fatal infection triggers expression of inflammasome components, activates caspase-1 and caspase-11, and induces host-cell death and secretion of IL-1β, IL-1α, and type I interferon (IFN-I). Wild-type and Casp1^−/− mice were highly susceptible to fatal ehrlichiosis, had overwhelming infection, and developed extensive tissue injury. Nlrp3^−/− mice effectively cleared ehrlichiae, but displayed acute mortality and developed liver injury similar to wild-type mice. By contrast, Ifnar1^−/− mice were highly resistant to fatal disease and had lower bacterial burden, attenuated pathology, and prolonged survival. Ifnar1^−/− mice also had improved protective immune responses mediated by IFN-γ and CD4⁺ Th1 and natural killer T cells, with lower IL-10 secretion by T cells. Importantly, heightened resistance of Ifnar1^−/− mice correlated with improved autophagosome processing, and attenuated noncanonical inflammasome activation indicated by decreased activation of caspase-11 and decreased IL-1β, compared with other groups. Our findings demonstrate that IFN-I signaling promotes host susceptibility to fatal ehrlichiosis, because it mediates ehrlichia-induced immunopathology and supports bacterial replication, perhaps via activation of noncanonical inflammasomes, reduced autophagy, and suppression of protective CD4⁺ T cells and natural killer T-cell responses against ehrlichiae.Ehrlichia chaffeensis is the causative agent of human monocytotropic ehrlichiosis, a highly prevalent life-threatening tickborne disease in North America.^{1, 2, 3} Central to the pathogenesis of human monocytotropic ehrlichiosis is the ability of ehrlichiae to survive and replicate inside the phagosomal compartment of host macrophages and to secrete proteins via type I and type IV secretion systems into the host-cell cytosol.⁴ Using murine models of ehrlichiosis, we and others have demonstrated that fatal ehrlichial infection is associated with severe tissue damage caused by TNF-α–producing cytotoxic CD8⁺ T cells (ie, immunopathology) and the suppression of protective CD4⁺ Th1 immune responses.^{5, 6, 7, 8, 9, 10, 11, 12, 13, 14} However, neither how the Ehrlichia bacteria trigger innate immune responses nor how these responses influence the acquired immunity against ehrlichiae is entirely known.Extracellular and intracellular pattern recognition receptors recognize microbial infections.^{15, 16, 17, 18} Recently, members of the cytosolic nucleotide-binding domain and leucine-rich repeat family (NLRs; alias NOD-like receptors), such as NLRP3, have emerged as critical pattern recognition receptors in the host defense against intracellular pathogens. NLRs recognize intracellular bacteria and trigger innate, protective immune responses.^{19, 20, 21, 22, 23} NLRs respond to both microbial products and endogenous host danger signals to form multimeric protein platforms known as inflammasomes. The NLRP3 inflammasome consists of multimers of NLRP3 that bind to the adaptor molecules and apoptosis-associated speck-like protein (ASC) to recruit pro–caspase-1 and facilitate cleavage and activation of caspase-1.^{15, 16, 24} The canonical inflammasome pathway involves the cleavage of immature forms of IL-1β and IL-18 (pro–IL-1β and pro–IL-18) into biologically active mature IL-1β and IL-18 by active caspase-1.^{25, 26, 27, 28} The noncanonical inflammasome pathway marked by the activation of caspase-11 has been described recently. Active caspase-11 promotes the caspase-1–dependent secretion of IL-1β/IL-18 and mediates inflammatory lytic host-cell death via pyroptosis, a process associated with the secretion of IL-1α and HMGB1.^{17, 29, 30, 31} Several key regulatory checkpoints ensure the proper regulation of inflammasome activation.^{16, 32} For example, blocking autophagy by the genetic deletion of the autophagy regulatory protein ATG16L1 increases the sensitivity of macrophages to the inflammasome activation induced by TLRs.³³ Furthermore, TIR domain-containing adaptor molecule 1 (TICAM-1; alias TRIF) has been linked to inflammasome activation via the secretion of type I interferons α and β (IFN-α and IFN-β) and the activation of caspase-11 during infections with Gram-negative bacteria.^{2, 34, 35, 36, 37, 38, 39}We have recently demonstrated that fatal ehrlichial infection induces excess IL-1β and IL-18 production, compared with mild infection,^{8, 12, 13, 14} and that lack of IL-18 signaling enhances resistance of mice to fatal ehrlichiosis.¹² These findings suggest that inflammasomes play a detrimental role in the host defense against ehrlichial infection. Elevated production of IL-1β and IL-18 in fatal ehrlichiosis was associated with an increase in hepatic expression of IFN-α.¹⁴ IFN-I plays a critical role in the host defense against viral and specific bacterial infections.^{28, 36, 37, 40, 41, 42, 43} However, the mechanism by which type I IFN contributes to fatal ehrlichial infection remains unknown. Our present results reveal, for the first time, that IFNAR1 promotes detrimental inflammasome activation, mediates immunopathology, and impairs protective immunity against ehrlichiae via mechanisms that involve caspase-11 activation, blocking of autophagy, and production of IL-10. Our novel finding that lipopolysaccharide (LPS)-negative ehrlichiae trigger IFNAR1-dependent caspase-11 activation challenges the current paradigm that implicates LPS as the major microbial ligand triggering the noncanonical inflammasome pathway during Gram-negative bacterial infection.     

Commensal Bacterial Endocytosis in Epithelial Cells Is Dependent on Myosin Light Chain Kinase–Activated Brush Border Fanning by Interferon-γ

Abnormal bacterial adherence and internalization in enterocytes have been documented in Crohn disease, celiac disease, surgical stress, and intestinal obstruction and are associated with low-level interferon (IFN)-γ production. How commensals gain access to epithelial soma through densely packed microvilli rooted on the terminal web (TW) remains unclear. We investigated molecular and ultrastructural mechanisms of bacterial endocytosis, focusing on regulatory roles of IFN-γ and myosin light chain kinase (MLCK) in TW myosin phosphorylation and brush border fanning. Mouse intestines were sham operated on or obstructed for 6 hours by loop ligation with intraluminally administered ML-7 (a MLCK inhibitor) or Y27632 (a Rho-associated kinase inhibitor). After intestinal obstruction, epithelial endocytosis and extraintestinal translocation of bacteria were observed in the absence of tight junctional damage. Enhanced TW myosin light chain phosphorylation, arc formation, and brush border fanning coincided with intermicrovillous bacterial penetration, which were inhibited by ML-7 and neutralizing anti–IFN-γ but not Y27632. The phenomena were not seen in mice genetically deficient for long MLCK-210 or IFN-γ. Stimulation of human Caco-2BBe cells with IFN-γ caused MLCK-dependent TW arc formation and brush border fanning, which preceded caveolin-mediated bacterial internalization through cholesterol-rich lipid rafts. In conclusion, epithelial MLCK-activated brush border fanning by IFN-γ promotes adherence and internalization of normally noninvasive enteric bacteria. Transcytotic commensal penetration may contribute to initiation or relapse of chronic inflammation.Commensal bacteria, estimated at 100 trillion (10¹⁴), are normally confined to the gut lumen and not in direct contact with epithelial cells. However, abnormal bacterial adherence and internalization in enterocytes have been documented in patients and experimental models with Crohn disease,^1,2 celiac disease,^3,4 chronic psychological stress,⁵ surgical manipulation,⁶ and intestinal obstruction (IO).⁷ Recent in vitro studies have found transcellular passage of nonpathogenic, noninvasive bacteria in epithelial monolayers after exposure to inflammatory and metabolic stress, such as interferon (IFN)-γ,^8,9 hypoxia,¹⁰ and mitochondrial damage.¹¹ To date, the molecular mechanisms of bacterial endocytosis in enterocytes remain unclear.Polarized intestinal epithelial cells are endowed with densely packed apical microvilli or brush border (BB), which act as an ultrastructural barrier that impedes physical contact between enteric microbes and cellular soma.¹² However, recent findings have revealed that commensal bacteria are internalized into epithelial cells via cholesterol-rich lipid rafts situated at invaginations of apical membrane between adjacent microvilli.^8,13,14 It is still poorly understood how microbes of 0.5 to 1 μm gain access to the base of the intermicrovillous cleft. Actin-cored microvilli are rooted in the filamentous meshwork of the terminal web (TW), which consists of multiple proteins, including actin, myosin, fodrin, and spectrin.¹⁵ Early reports revealed that the phosphorylation of myosin light chain (MLC) in the TW region, via unknown kinases, leads to BB fanning in enterocytes.^16,17 Other studies have found that the phosphorylation of perijunctional MLC by myosin light chain kinase (MLCK) and Rho-associated kinase (ROCK) is involved in epithelial tight junction (TJ) disruption.^18,19 We hypothesized that TW MLC contraction and BB fanning may allow bacterial penetration through an enlarged intermicrovillous cleft to initiate apical endocytosis. The roles of MLCK and ROCK in mechanisms of bacterial endocytosis and their correlation with TJ changes have yet to be determined.Previous studies from our laboratory found increased bacterial translocation (BT) to extraintestinal organs after IO by loop ligation.^20,21 The current aim was to investigate molecular and ultrastructural mechanisms of apical bacterial endocytosis in enterocytes of IO models, focusing on the role of MLCK-dependent TW myosin phosphorylation and BB fanning. The regulatory role of IFN-γ was also examined using genetically deficient mice and epithelial cell cultures.     

CD19 Expression in B Cells Regulates Atopic Dermatitis in a Mouse Model

Atopic dermatitis is an inflammatory cutaneous disorder characterized by dry skin and relapsing eczematous skin lesions. Besides antibody production, the contribution of B cells to the pathogenesis of atopic dermatitis is unclear. In mice, repeated epicutaneous sensitization with ovalbumin induces inflamed skin lesions resembling human atopic dermatitis and therefore serves as an experimental model for this condition. To investigate the role of B cells in a murine model of atopic dermatitis, ovalbumin-sensitized allergic skin inflammation was assessed in mice lacking CD19. In ovalbumin-sensitized skin from CD19-deficient mice, the number of eosinophils and CD4⁺ T cells was reduced, and both epidermal and dermal thickening were decreased. Following in vitro stimulation with ovalbumin, CD19 deficiency significantly reduced the proliferation of CD4⁺, but not CD8⁺, T cells from spleen and draining lymph nodes. Furthermore, splenocytes and draining lymph node cells from ovalbumin-sensitized CD19-deficient mice secreted significantly less IL-4, IL-13, and IL-17 than ovalbumin-sensitized wild-type mice. These results suggest that CD19 expression in B cells plays a critical role in antigen-specific CD4⁺ T-cell proliferation and T helper 2 and 17 responses in a murine model of atopic dermatitis. Furthermore, the present findings may have implications for B-cell–targeted therapies for the treatment of atopic dermatitis.Atopic dermatitis (AD) is one of the most common inflammatory cutaneous disorders, characterized by dry, itchy skin and relapsing eczematous skin lesions, which affects approximately 15% to 30% of children and 2% to 10% of adults.¹ Histologically, AD is characterized by epidermal and dermal thickening with marked infiltration of activated T cells, eosinophils, and monocytes/macrophages within the dermis.¹ Approximately 60% to 90% of patients with AD show increased serum total IgE against environmental and/or food allergens.^2–4 In addition, the expression of T helper (Th) 2 cytokines, such as IL-4, IL-5, and IL-13, is increased in the acute skin lesions of AD,^5,6 suggesting that Th2 cells play critical roles in disease development.Skin barrier dysfunction is a critical feature of AD. Recent studies have shown that more than 10% of patients with AD have mutations in the filaggrin gene, which is important for skin barrier function.^7,8 It has been hypothesized that a disrupted skin barrier facilitates antigen penetration and epicutaneous sensitization, leading to allergic skin inflammation in patients with AD.⁹ Moreover, IL-4 and IL-13 reduce filaggrin gene and protein expression in keratinocytes.¹⁰ Thus, a genetic and/or acquired defect in filaggrin is likely to play an important role in the development of AD. In mice, repeated epicutaneous sensitization of tape-stripped skin with ovalbumin (OVA), mimicking epicutaneous allergen exposure to epidermal barrier dysfunction, was found to induce the appearance of inflamed pruritic skin lesions at the application site, as well as local and systemic Th2 responses. Because of the resemblance of these lesions to human AD,^11,12 this experimental method can serve as a convenient experimental model.Historically, B cells have been considered to mediate humoral immune responses by differentiating into antibody (Ab)-secreting plasma cells.¹³ However, recent studies have revealed that B cells also serve as antigen-presenting cells,¹⁴ secrete a variety of cytokines,¹⁵ provide costimulatory signals, and promote T-cell activation.^15,16 Moreover, IL-10–producing B cell subsets can inhibit innate and adaptive immune responses, inflammation, and autoimmunity, demonstrating the existence of regulatory B cells.^13,17–19 Thus, in addition to Ab production, B cells have multiple diverse immune functions.The fate and function of B cells are controlled by signal transduction through B-cell receptors, which are further modified by other cell-surface molecules, including CD19, CD21, CD22, CD40, CD72, and Fcγ receptor IIb.²⁰ CD19 is a general rheostat that defines signaling thresholds critical for humoral immune responses and autoimmunity.²¹ CD19 is a B-cell–specific cell-surface molecule of the Ig superfamily expressed by early pre-B cells in humans and mice until plasma cell differentiation.^22,23 Human CD19 and mouse CD19 are functionally equivalent in vivo.²² B cells from CD19-deficient (CD19^−/−) mice are hyporesponsive to a variety of transmembrane signals, including B-cell receptor ligation.²² CD20, a B-cell–specific cell-surface molecule involved in the regulation of B-cell activation and Ca²⁺ transport, is initially expressed by pre-B cells in humans and mice with continued expression until plasma cell differentiation.^24,25 Although the role of B cells, besides Ab production, in the pathogenesis of AD remains unclear, B-cell depletion in humans with the chimeric human anti-CD20 monoclonal antibody (mAb) rituximab results in an improvement of AD,^26,27 suggesting that B cells play important roles in the development of this condition. Therefore, in the present study, we examined the importance of B cells in an OVA-sensitized allergic skin inflammation model using CD19^−/− and wild-type (WT) mice.     

Loss of β-Catenin Induces Multifocal Periosteal Chondroma-Like Masses in Mice

Osteochondromas and enchondromas are the most common tumors affecting the skeleton. Osteochondromas can occur as multiple lesions, such as those in patients with hereditary multiple exostoses. Unexpectedly, while studying the role of β-catenin in cartilage development, we found that its conditional deletion induces ectopic chondroma-like cartilage formation in mice. Postnatal ablation of β-catenin in cartilage induced lateral outgrowth of the growth plate within 2 weeks after ablation. The chondroma-like masses were present in the flanking periosteum by 5 weeks and persisted for more than 6 months after β-catenin ablation. These long-lasting ectopic masses rarely contained apoptotic cells. In good correlation, transplants of β-catenin-deficient chondrocytes into athymic mice persisted for a longer period of time and resisted replacement by bone compared to control wild-type chondrocytes. In contrast, a β-catenin signaling stimulator increased cell death in control chondrocytes. Immunohistochemical analysis revealed that the amount of detectable β-catenin in cartilage cells of osteochondromas obtained from hereditary multiple exostoses patients was much lower than that in hypertrophic chondrocytes in normal human growth plates. The findings in our study indicate that loss of β-catenin expression in chondrocytes induces periosteal chondroma-like masses and may be linked to, and cause, the persistence of cartilage caps in osteochondromas.Osteochondromas and enchondromas are the most common tumors affecting the skeleton.^1,2 Osteochondromas are cartilage-covered masses that form near the growth plate and bone surface, whereas enchondromas form within the growth plate and bone marrow. Both types of benign tumors can cause mechanical impairment of movement and also pain due to impingement or compression of nerves and blood vessels, particularly when they are present at multiple sites.^3,4 These benign tumors may become malignant.^5–7 The potential for malignant progression is greater in patients with syndromes, such as Ollier disease, Maffucci syndrome, or hereditary multiple exostoses (HME), the latter also known as multiple osteochondroma.^5–7 Current treatments largely rely on surgical excision.^3,8 Both benign and malignant cartilage tumors are generally resistant to chemotherapy and radiotherapy.^5,9 Thus, a better understanding of the cellular and molecular mechanisms underlying cartilage tumor formation and growth is critical for the development of new therapeutic strategies and treatments.Recent studies have indicated that several genes play important roles in cartilage tumor formation.^4,5 Hopyan et al¹⁰ found mutations in parathyroid hormone receptor 1 (PTHR1) in patients with multiple enchondromas. These authors generated mice harboring the same PTHR1 mutations that displayed a similar enchondroma formation. In addition, they found that the PTHR1 mutations caused constitutive activation of hedgehog signaling in cultured chondrocytes and that overexpression of Gli2, a downstream molecule of hedgehog signaling, induced enchondromas in mice.¹⁰ In the follow-up studies, however, it was found that certain cohorts of enchondromatosis patients do not have PTHR1 mutations¹¹ and that enchondroma formation may actually be independent of hedgehog signaling.¹² Thus, the pathogenesis of enchondroma formation remains to be clarified.Mutations in EXT1 and EXT2 genes have been associated with hereditary multiple exostoses (HME) (multiple osteochondroma).^5–7 Mutations in these genes are often missense or frame shift and cause synthesis of lower levels of (and shorter) heparan sulfate chains.^4,5 This is because EXT1 and EXT2 encode Golgi-associated enzymes are responsible for the polymerization of the chains.¹³ Insufficiency of heparan sulfate-rich proteoglycans is thought to be a cause of osteochondroma formation.^4,5,13 Heparan sulfate proteoglycans are important for the regulation of many signaling pathways that include hedgehog, bone morphogenetic protein, fibroblast growth factor, and Wnt pathways.^13,14 All of these pathways are critical regulators for chondrogenic differentiation and chondrocyte differentiation.^15,16 It is likely that dysregulation of these signaling pathways resulting from heparan sulfate deficiency may trigger abnormal behavior of growth plate chondrocytes or induce ectopic chondrogenic differentiation, leading to ectopic cartilage formation. Recently, several Ext mutant mouse lines have been established.^17–19 All of these transgenic mouse lines show multifocal ectopic cartilaginous masses with microscopical and structural similarities to osteochondromas found in HME patients.^17–19 The cellular and molecular mechanisms underlying EXT mutation-associated chondroma formation, however, remains largely unclear.The Wnt/β-catenin signaling pathway is essential for regulation of normal cartilage development, maintenance of permanent cartilage, and growth plate function.^20–24 Previous reports have shown that inactivation of this signaling pathway impairs cartilage and skeletal development. Conditional ablation of the β-catenin gene in limb skeletogenic cells induces a delay in endochondral bone formation and the formation of abnormal cartilaginous masses during embryonic development.^25–27 In addition, overexpression of a Wnt antagonist strongly inhibits both hypertrophy of chondrocytes and progression of endochondral ossification.²⁸ Recently, we generated compound transgenic mice in which we induced postnatal conditional ablation of β-catenin in cartilage.²⁴ We found that the resulting β-catenin deficiency impaired growth plate function and skeletal growth. In addition, the mice developed ectopic cartilaginous masses located near the bone surface but not within the bone marrow. In the present study, we characterized the pathohistology of these ectopic cartilaginous masses and investigated their possible cell origin and fate, and also related the findings to human osteochondromas.     

p120-Catenin Expressed in Alveolar Type II Cells Is Essential for the Regulation of Lung Innate Immune Response

The integrity of the lung alveolar epithelial barrier is required for the gas exchange and is important for immune regulation. Alveolar epithelial barrier is composed of flat type I cells, which make up approximately 95% of the gas-exchange surface, and cuboidal type II cells, which secrete surfactants and modulate lung immunity. p120-catenin (p120; gene symbol CTNND1) is an important component of adherens junctions of epithelial cells; however, its function in lung alveolar epithelial barrier has not been addressed in genetic models. Here, we created an inducible type II cell–specific p120-knockout mouse (p120EKO). The mutant lungs showed chronic inflammation, and the alveolar epithelial barrier was leaky to ¹²⁵I-albumin tracer compared to wild type. The mutant lungs also demonstrated marked infiltration of inflammatory cells and activation of NF-κB. Intracellular adhesion molecule 1, Toll-like receptor 4, and macrophage inflammatory protein 2 were all up-regulated. p120EKO lungs showed increased expression of the surfactant proteins Sp-B, Sp-C, and Sp-D, and displayed severe inflammation after pneumonia caused by Pseudomonas aeruginosa compared with wild type. In p120-deficient type II cell monolayers, we observed reduced transepithelial resistance compared to control, consistent with formation of defective adherens junctions. Thus, although type II cells constitute only 5% of the alveolar surface area, p120 expressed in these cells plays a critical role in regulating the innate immunity of the entire lung.Lungs are constantly exposed to pathogens; therefore, a highly restrictive alveolar epithelial barrier and finely tuned host defense mechanisms are indispensable for their protection.^1,2 Unchecked inflammation is linked to various acute and chronic diseases, including edema, acute respiratory distress syndrome, and fibrosis.^3,4 Although it is abundantly clear that the alveolar epithelial barrier regulates the transport of gases, liquid, and ions,^5,6 the role of the barrier in the regulation of the innate immune function of lungs remains poorly understood.The restrictiveness of the alveolar epithelial barrier is dependent on a series of interacting proteins comprising the adherens junctions (AJs) and tight junctions (TJs).^7,8 The core of the epithelial AJs is composed of E-cadherin, which links cells to one another in the monolayer.⁹ The cytoplasmic domain of E-cadherin associates with α-catenin, β-catenin, and p120-catenin (p120, official name catenin delta 1; CTNND1).⁹ The α- and β-catenins can recruit proteins that link E-cadherin to the actin cytoskeleton,⁹ and together, these interactions maintain the tension landscape in the epithelial monolayer.¹⁰ β-Catenin also plays an essential role in the Wnt signaling pathway and thereby contributes to cell proliferation and differentiation.¹¹ However, p120 has received comparatively less attention, although recent studies have shown that p120 has important functions in regulating cadherin stability and turnover¹² and innate immunity.¹³Here, we focused on the role of p120 expressed in alveolar epithelial type II cells in regulating the innate immune function of lungs. Although alveolar type II cells cover only 5% of the alveolar surface area, these cells are metabolically active.¹⁴ They produce surfactants, serve as facultative progenitor cells to repair alveolar injury, and regulate innate immune function of the lung.¹⁴ These cells express Toll-like receptors (TLRs) and tumor necrosis factor receptors.¹⁵ Interactions with pathogens or endotoxins activate these receptors to initiate NF-κB signaling to produce tumor necrosis factor,¹⁶ IL-1 and IL-6,¹⁶ regulated on activation normal T cell expressed and secreted,¹⁷ and chemokine C-X-C motif ligand 1.¹⁸ These factors play key roles in recruiting inflammatory cells.^19–21 Alveolar type II cells also secrete the surfactant proteins (Sp)-A, -B, -C, and -D,²² which regulate innate and adaptive immunity by binding to antigen through interactions with surface receptors on inflammatory cell membranes.²³ Here, we studied the function of p120 through disrupting the p120 gene in alveolar type II cells in mice using the rtTA/TetO system coupled with a type II cell–specific SPC promoter. In these mice, we observed unchecked chronic lung inflammation associated with increased NF-κB activity and a persistently leaky alveolar epithelial barrier. These results provide the first genetic evidence that p120 in type II cells is a central regulator of innate immunity of lungs.