Lipopolysaccharide Causes an Increase in Intestinal Tight Junction Permeability in Vitro and in Vivo by Inducing Enterocyte Membrane Expression and Localization of TLR-4 and CD14

Bacterial-derived lipopolysaccharides (LPS) play an essential role in the inflammatory process of inflammatory bowel disease. A defective intestinal tight junction (TJ) barrier is an important pathogenic factor of inflammatory bowel disease and other inflammatory conditions of the gut. Despite its importance in mediating intestinal inflammation, the physiological effects of LPS on the intestinal epithelial barrier remain unclear. The major aims of this study were to determine the effects of physiologically relevant concentrations of LPS (0 to 1 ng/mL) on intestinal barrier function using an in vitro (filter-grown Caco-2 monolayers) and an in vivo (mouse intestinal perfusion) intestinal epithelial model system. LPS, at physiologically relevant concentrations (0 to 1 ng/mL), in the basolateral compartment produced a time-dependent increase in Caco-2 TJ permeability without inducing cell death. Intraperitoneal injection of LPS (0.1 mg/kg), leading to clinically relevant plasma concentrations, also caused a time-dependent increase in intestinal permeability in vivo. The LPS-induced increase in intestinal TJ permeability was mediated by an increase in enterocyte membrane TLR-4 expression and a TLR-4–dependent increase in membrane colocalization of membrane-associated protein CD14. In conclusion, these studies show for the first time that LPS causes an increase in intestinal permeability via an intracellular mechanism involving TLR-4–dependent up-regulation of CD14 membrane expression.An integral function of intestinal epithelial cells is to act as a physical barrier, separating the noxious luminal environment from the underlying lamina propria and the deeper intestinal layers.^1,2 The apically located tight junctions (TJs) form a paracellular seal between the lateral membranes of adjacent intestinal epithelial cells, and act as a structural and functional barrier against paracellular flux of luminal substances. Defective intestinal epithelial TJ barrier has been shown to be an important pathogenic factor of inflammatory bowel disease (IBD) and necrotizing enterocolitis (NEC) by allowing paracellular permeation of luminal antigens that elicit and promote inflammatory response.^1,2 Both clinical and animal studies have shown the importance of a defective intestinal TJ barrier in the development and prolongation of intestinal inflammation in IBD and NEC.^1–5 These studies have shown that normalization of intestinal barrier in patients with active Crohn’s disease predicts prolonged clinical remission, whereas a persistent increase in intestinal permeability portends poor clinical outcome with rapid recurrence of the disease.^6,7 Additionally, animal studies have also shown that a primary defect in intestinal junctional complexes was sufficient to induce or aggravate intestinal inflammation in murine models of IBD,^8,9 whereas therapeutic tightening or enhancement of the intestinal TJ barrier prevented the development of intestinal inflammation.^3,10The terms endotoxin and lipopolysaccharide (LPS) are used interchangeably and refer to the major cell wall component of Gram-negative bacteria.^11,12 LPS are complex amphiphilic molecules having a hydrophobic (consisting of lipid A) and a hydrophilic (consisting of carbohydrate core and polysaccharide O-antigen) component and are released from bacterial cell wall by shedding or through bacterial lysis.^11–13 LPS concentrations are highest in the gut lumen, where many trillions of commensal bacteria reside. Normally, LPS in the gut lumen do not penetrate across the healthy intestinal epithelium^14,15; however, in intestinal permeability disorders, the defective TJ barrier allows paracellular flux of LPS and other luminal antigens.^{11–13,16–19} The intestinal tissue and circulating LPS levels are markedly elevated in IBD and NEC, and play an important role in mediating inflammatory response.^{11–13,16–18} The involvement of LPS in the initiation and propagation of intestinal inflammation in IBD and NEC has been well demonstrated.^20–23 These studies have shown LPS to be an important contributing factor of intestinal inflammation, and removal of circulating LPS accelerated the clinical improvement of IBD and NEC.^20,22,23 Despite the importance of a defective intestinal barrier in the accentuation and prolongation of intestinal inflammation in IBD and NEC,^3,6,9,20,22 the effects of circulating levels of LPS on the intestinal epithelial barrier remain unknown. Because LPS levels are markedly elevated in these diseases and play an important role in the inflammatory process, understanding the effects of LPS on intestinal barrier function has important potential clinical significance.In normal healthy individuals, plasma concentrations of LPS range from undetectable levels up to 0.2 ng/mL.^11,12,20,22 A variety of physiological factors such as prolonged physical exertion, high-fat diet, physiological stresses, or heat can lead to elevated plasma LPS levels as high as 1 to 2 ng/mL.^24–27 Patients with intestinal permeability disorders such as Crohn’s disease, NEC, acute pancreatitis, alcoholic liver disease, and critical illnesses also have elevated plasma LPS levels ranging up to 2 to 10 ng/mL.^{11–13,20,22,28} Based on these reports, we consider LPS levels of 0 to 1.0 ng/mL to be physiologically relevant and 0 to 10 ng/mL to be clinically relevant. (For reference, the concentration of LPS in the gut lumen has been reported to be 1.8 μg/mL in the rat distal ileum.^29,30) Inexplicably, in most of the published studies, extreme pharmacological concentrations of LPS ranging between 50 and 1000 μg/mL, which exceed the physiologically achievable concentrations by 10⁴- to 10⁷-fold, have been used to assess various biological responses.^30–34 At these extreme concentrations, LPS causes rapid cell death in various cell types studied, including in intestinal and immune cells,^30,33–35 and does not provide accurate depiction of biological activity of LPS. Herein, we show that LPS, at physiologically and clinically relevant concentrations (0 to 10 ng/mL), does not cause intestinal epithelial cell death, but causes a selective increase in intestinal TJ permeability in vitro and in vivo. These studies also show for the first time that pattern recognition receptors Toll-like receptor 4 (TLR-4) and CD14 play a central role in the modulation of the intestinal epithelial TJ barrier.     

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.     

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.     

Loss of Cxcr4 in Endometriosis Reduces Proliferation and Lesion Number while Increasing Intraepithelial Lymphocyte Infiltration

Hyperactivation of the CXCL12-CXCR4 axis occurs in endometriosis; the therapeutic potential of treatments aimed at global inhibition of the axis was recently reported. Because CXCR4 is predominantly expressed on epithelial cells in the uterus, this study explored the effects of targeted disruption of CXCR4 in endometriosis lesions. Uteri derived from adult female mice homozygous for a floxed allele of CXCR4 and co-expressing Cre recombinase under control of progesterone receptor promoter were sutured onto the peritoneum of cycling host mice expressing the green fluorescent protein. Four weeks after endometriosis induction, significantly lower number of lesions developed in Cxcr4-conditional knockout lesions relative to those in controls (37.5% vs. 68.8%, respectively). In lesions that developed in Cxcr4-knockout, reduced epithelial proliferation was associated with a lower ratio of epithelial to total lesion area compared with controls. Furthermore, while CD3⁺ lymphocytes were largely excluded from the epithelial compartment in control lesions, in Cxcr4-knockout lesions, CD3⁺ lymphocytes infiltrated the Cxcr4-deficient epithelium in the diestrus and proestrus stages. Current data demonstrate that local CXCR4 expression is necessary for proliferation of the epithelial compartment of endometriosis lesions, that its downregulation compromises lesion numbers, and suggest a role for epithelial CXCR4 in lesion immune evasion.

Endometriosis is one of the most common gynecologic diseases in women of reproductive age, with a prevalence rate of approximately 10%.¹ Various theories have been proposed for the origin of endometriosis, including the most widely accepted theory of retrograde menstruation, in which shed endometrial tissue is refluxed through the fallopian tubes and proliferates within the pelvis.^2,3 Because the majority of women have retrograde menstruation, yet only about one in 10 develops endometriosis, it has been proposed that factors promoting survival, invasiveness, and growth of endometrial fragments in the peritoneal cavity play a role in their persistence at ectopic sites in women with endometriosis. Such predisposing factors include somatic mutations in the highly proliferative endometrial epithelium (ie, KRAS, ARID1A⁴), aberrant progenitor/stem cell populations (endometrial or bone marrow (BM) derived5, 6, 7, 8, 9) at ectopic sites, and/or an immune-tolerant microenvironment permissive to proliferation and neoangiogenesis of ectopic endometrial fragments. This immunosuppressive microenvironment is characterized by elevated levels of activated peritoneal macrophages, reduced natural killer cell activity, and abnormally high levels of T-regulatory cells,¹⁰ which suppress immune mechanisms aimed at eliminating implantation of misplaced autologous cells.The chemokine-receptor CXCL12-CXCR4 axis is up-regulated in endometriosis.11, 12, 13, 14 The axis has roles in promoting cell survival, proliferation, chemotaxis, invasion, and angiogenesis. In cancer, hyperactivation of the axis is associated with disease progression and correlates with poor clinical outcome.15, 16, 17, 18, 19 This axis was also proposed to function in immune modulation: CXCL12 binding to CXCR4-expressing intratumoral (epithelial) cells was suggested to be a mechanism mediating cancer evasion of immune surveillance.^20,21 Therapeutic blockade of the axis with the CXCR4 antagonist AMD3100 exhibited antitumor effects, including reduced tumor proliferation and increased apoptosis, both associated with T-cell accumulation within the tumor epithelium.^20,22, 23, 24Endometrial CXCR4 is predominantly expressed on luminal and glandular epithelia, whereas the stroma is the principal source of the ligand CXCL12.^13,25 Stromal-derived CXCL12 exerts its proliferative effect on the epithelium through paracrine interactions with its cognate receptor CXCR4.²⁶ Estradiol stimulates CXCL12 production and progesterone to inhibit this stimulation.^27,28 In vitro, AMD3100 blocked the CXCL12-mediated proliferative effects on epithelial cells.²⁹ Acute treatment of experimental endometriosis in mice with AMD3100 significantly decreases lesion volume and reduces BM cell trafficking to lesions.³⁰ AMD3100 was also shown to reduce recruitment of BM-derived endothelial progenitor cells into lesions and compromise their vascularization.³¹ Based on these studies, whether the inhibitory action of AMD3100 on lesion growth is mediated via local effects (ie, inhibiting lesion-endogenous CXCR4) or systemic effects (ie, inhibiting exogenous CXCR4-expressing cells, which infiltrate lesions with endometriosis induction) was explored. Moreover, in a manner similar to cancer, lesion-derived CXCR4 may have a role in immune evasion.To achieve these goals, endometriosis was induced using uteri derived from 8- to 10- week–old Pgr^Cre/+ Cxcr4^−/− female mice homozygous for a floxed CXCR4 allele and harboring a progesterone receptor promoter–driven Cre recombinase. Endometriosis was induced in syngeneic green fluorescent protein (GFP) transgenic host mice, allowing discrimination of host-derived populations from endometrial cells within uterine explants. A significant reduction in the number of lesions was found in mice harboring Cxcr4-conditional knockout lesions. In lesions that did develop, epithelial thinning was observed concomitant with the appearance of intraepithelial lymphocytes. At the proliferative stage, Ki-67 staining was absent from the epithelium of lesions, suggesting that diminished lesion numbers may be attributed to loss of epithelial proliferation, ultimately undermining lesion integrity.     

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.     

Sebaceous Gland,Hair Shaft,and Epidermal Barrier Abnormalities in Keratosis Pilaris with and without Filaggrin Deficiency

Although keratosis pilaris (KP) is common, its etiopathogenesis remains unknown. KP is associated clinically with ichthyosis vulgaris and atopic dermatitis and molecular genetically with filaggrin-null mutations. In 20 KP patients and 20 matched controls, we assessed the filaggrin and claudin 1 genotypes, the phenotypes by dermatoscopy, and the morphology by light and transmission electron microscopy. Thirty-five percent of KP patients displayed filaggrin mutations, demonstrating that filaggrin mutations only partially account for the KP phenotype. Major histologic and dermatoscopic findings of KP were hyperkeratosis, hypergranulosis, mild T helper cell type 1-dominant lymphocytic inflammation, plugging of follicular orifices, striking absence of sebaceous glands, and hair shaft abnormalities in KP lesions but not in unaffected skin sites. Changes in barrier function and abnormal paracellular permeability were found in both interfollicular and follicular stratum corneum of lesional KP, which correlated ultrastructurally with impaired extracellular lamellar bilayer maturation and organization. All these features were independent of filaggrin genotype. Moreover, ultrastructure of corneodesmosomes and tight junctions appeared normal, immunohistochemistry for claudin 1 showed no reduction in protein amounts, and molecular analysis of claudin 1 was unremarkable. Our findings suggest that absence of sebaceous glands is an early step in KP pathogenesis, resulting in downstream hair shaft and epithelial barrier abnormalities.Keratosis pilaris (KP) is a disorder of keratinization, characterized by prominent keratinous plugging of follicular orifices, and various degrees of perifollicular erythema.^1–3 Although the proximal extremities are a common area of involvement, other sites of predilection include the buttocks, trunk, and face.^1,2 The onset of KP usually occurs during the first two decades of life with a peak at puberty,⁴ followed by either improvement with increasing age in 35%, persistence into adulthood in 43%, or worsening in 22% of the patients.⁵ Prevalence estimates for KP range between 4% and 34%,^6,7 and earlier studies report a prevalence up to 80% in British girls,⁸ demonstrating that the KP phenotype is common.Although family studies suggest an autosomal dominant pattern of inheritance,⁵ no single gene mutation has yet been linked to KP. KP is associated with atopic dermatitis in large population samples,^1,4,5,7 ichthyosis vulgaris, and hence filaggrin (FLG) mutations.^7,9–12The clinical expression of KP is likely determined not only by genetic predisposition but also by environmental factors. Some persons with KP experience improvement during the summer, compared with winter months, suggesting a role for reduced environmental humidity in disease expression.^4,5 A stronger association was found between FLG mutations and KP in populations residing in colder and drier temperate zones than in equatorial populations.¹³ An acquired increase in the incidence of KP-like lesions is observed in obesity, diabetes, pregnancy, menopause, and malnutrition, particularly in association with vitamin A deficiency.^14–18 Treatment of KP has focused on preventing excessive skin dryness with moisturizers, softening and thinning the follicular plugs with keratolytic agents, peelings, or topical retinoids, and/or decreasing associated erythema with low-potency topical steroids.^2,19The pathogenesis of KP remains unknown, and the literature about this topic is limited. Because both ichthyosis vulgaris and atopic dermatitis are strongly associated with KP,^9–11 it is possible that the follicular abnormalities observed in KP result from FLG deficiency. FLG, one major structural protein of the epidermis, not only aggregates keratin filaments into corneocytes but also is hydrolyzed into osmotically active amino acids, forming approximately 50% of the natural moisturizing factor of the stratum corneum (SC), and playing a key role in photoprotection and acidifying the skin surface.^20,21 Null mutations in FLG result in reduced natural moisturizing factor in the SC, leading to xerosis cutis and epithelial barrier abnormality.^20,22,23In normal epidermis, the permeability barrier is formed by corneocytes surrounded by the hydrophobic extracellular lamellar bilayers of the SC, which derive from the secretion of lamellar body (LB) contents, with an additional potential role of tight junctions (TJs) of the subjacent stratum granulosum (SG).²⁴ Corneodesmosomes regulate the integrity and cohesion of the SC.²⁵ Together, these structures provide the epidermis with a formidable barrier against the outward loss of water and electrolytes, while also preventing transcutaneous entry of exogenous xenobiotics.^26–28We assessed here the FLG genotype of 20 KP patients and the morphology of KP skin by light and electron microscopy. Furthermore, we asked whether KP is associated with changes in epidermal structure and whether the putative barrier impairment is only observed in KP patients with FLG null mutations.     

Synergistic Actions of Blocking Angiopoietin-2 and Tumor Necrosis Factor-α in Suppressing Remodeling of Blood Vessels and Lymphatics in Airway Inflammation

Remodeling of blood vessels and lymphatics are prominent features of sustained inflammation. Angiopoietin-2 (Ang2)/Tie2 receptor signaling and tumor necrosis factor-α (TNF)/TNF receptor signaling are known to contribute to these changes in airway inflammation after Mycoplasma pulmonis infection in mice. We determined whether Ang2 and TNF are both essential for the remodeling on blood vessels and lymphatics, and thereby influence the actions of one another. Their respective contributions to the initial stage of vascular remodeling and sprouting lymphangiogenesis were examined by comparing the effects of function-blocking antibodies to Ang2 or TNF, given individually or together during the first week after infection. As indices of efficacy, vascular enlargement, endothelial leakiness, venular marker expression, pericyte changes, and lymphatic vessel sprouting were assessed. Inhibition of Ang2 or TNF alone reduced the remodeling of blood vessels and lymphatics, but inhibition of both together completely prevented these changes. Genome-wide analysis of changes in gene expression revealed synergistic actions of the antibody combination over a broad range of genes and signaling pathways involved in inflammatory responses. These findings demonstrate that Ang2 and TNF are essential and synergistic drivers of remodeling of blood vessels and lymphatics during the initial stage of inflammation after infection. Inhibition of Ang2 and TNF together results in widespread suppression of the inflammatory response.Remodeling of blood vessels and lymphatics contributes to the pathophysiology of many chronic inflammatory diseases, including asthma, chronic bronchitis, chronic obstructive pulmonary disease, inflammatory bowel disease, and psoriasis.^{1, 2, 3} When inflammation is sustained, capillaries acquire venule-like properties that expand the sites of plasma leakage and leukocyte influx. Consistent with this transformation, the remodeled blood vessels express P-selectin, intercellular adhesion molecule 1 (ICAM-1), EphB4, and other venular markers.^{4, 5, 6} The changes are accompanied by remodeling of pericytes and disruption of pericyte-endothelial crosstalk involved in blood vessel quiescence.⁷ Remodeling of blood vessels is accompanied by plasma leakage, inflammatory cell influx, and sprouting lymphangiogenesis.^{6, 8, 9}Mycoplasma pulmonis infection causes sustained inflammation of the respiratory tract of rodents.¹⁰ This infection has proved useful for dissecting the features and mechanisms of vascular remodeling and lymphangiogenesis.^{6, 9, 10} At 7 days after infection, there is widespread conversion of capillaries into venules, pericyte remodeling, inflammatory cell influx, and lymphatic vessel sprouting in the airways and lung.^{4, 5, 6, 7, 8, 9} Many features of chronic M. pulmonis infection in mice are similar to Mycoplasma pneumoniae infection in humans.¹¹Angiopoietin-2 (Ang2) is a context-dependent antagonist of Tie2 receptors^{12, 13} that is important for prenatal and postnatal remodeling of blood vessels and lymphatic vessels.^{13, 14, 15} Ang2 promotes vascular remodeling,^{4, 5} lymphangiogenesis,^{15, 16, 17} and pericyte loss¹⁸ in disease models in mice. Mice genetically lacking Ang2 have less angiogenesis, lymphangiogenesis, and neutrophil recruitment in inflammatory bowel disease.³ Ang2 has proved useful as a plasma biomarker of endothelial cell activation in acute lung injury, sepsis, hypoxia, and cancer.¹⁹Like Ang2, tumor necrosis factor (TNF)-α is a mediator of remodeling of blood vessels and lymphatics.^{8, 9, 20, 21} TNF triggers many components of the inflammatory response, including up-regulation of expression of vascular cell adhesion molecule-1, ICAM-1, and other endothelial cell adhesion molecules.²² TNF inhibitors reduce inflammation in mouse models of inflammatory disease^{23, 24} and are used clinically in the treatment of rheumatoid arthritis, ankylosing spondylitis, Crohn''s disease, psoriatic arthritis, and some other inflammatory conditions.^{24, 25} Indicative of the complex role of TNF in disease, inhibition or deletion of TNF can increase the risk of serious infection by bacterial, mycobacterial, fungal, viral, and other opportunistic pathogens.²⁶TNF and Ang2 interact in inflammatory responses. TNF increases Ang2 expression in endothelial cells in a time- and dose-dependent manner, both in blood vessels²⁷ and lymphatics.¹⁶ Administration of TNF with Ang2 increases cell adhesion molecule expression more than TNF alone.^{16, 28} Similarly, Ang2 can promote corneal angiogenesis in the presence of TNF, but not alone.²⁹ In mice that lack Ang2, TNF induces leukocyte rolling but not adherence to the endothelium.²⁸ Ang2 also augments TNF production by macrophages.^{30, 31} Inhibition of Ang2 and TNF together with a bispecific antibody can ameliorate rheumatoid arthritis in a mouse model.³²With this background, we sought to determine whether Ang2 and TNF act together to drive the remodeling of blood vessels and lymphatics in the initial inflammatory response to M. pulmonis infection. In particular, we asked whether Ang2 and TNF have synergistic actions in this setting. The approach was to compare the effects of selective inhibition of Ang2 or TNF, individually or together, and then assess the severity of vascular remodeling, endothelial leakiness, venular marker expression, pericyte changes, and lymphatic sprouting. Functional consequences of genome-wide changes in gene expression were analyzed by Ingenuity Pathway Analysis (IPA)^{33, 34} and the Database for Annotation, Visualization and Integrated Discovery (DAVID).³⁵ The studies revealed that inhibition of Ang2 and TNF together, but not individually, completely prevented the development of vascular remodeling and lymphatic sprouting and had synergistic effects in suppressing gene expression and cellular pathways activated during the initial stage of the inflammatory response.     

Reduced Innervation and Delayed Re-Innervation After Epithelial Wounding in Type 2 Diabetic Goto-Kakizaki Rats

Patients with diabetes are at an increased risk for developing corneal complications including delayed wound healing and potential vision loss. To understand the cause of diabetic keratopathy, we investigated innervation and its correlation with delayed corneal epithelial wound healing in type 2 diabetic Goto-Kakizaki (GK) rats. GK rats are smaller than the age-matched control Wistar rats from which the GK rats were derived. The blood sugar levels of GK rats are significantly higher than those of Wistar rats. GK rats had increased rose bengal staining and cornea fragility. Fewer nerve fibers were detected compared with Wistar rats. Although nerve fiber densities detected by whole-mount immunohistochemistry were similar near the limbal region, in the central cornea the subbasal nerve plexuses were thinner, less abundant, and showed less branching in GK rats. Corneal epithelial wound closure was delayed and re-innervation was slow and incomplete in GK rats. These abnormalities were more apparent in older GK rats (12 months). Our data suggest that diabetic neuropathy occurs in the cornea of type 2 diabetic GK rats, and defects in the sensory nerve and/or tear film may contribute to diabetic keratopathy and delayed epithelial wound healing in diabetic corneas.With the rapid increase in the prevalence of diabetes mellitus (DM), mostly in type 2 DM, ocular complications have become a leading cause of blindness in the world.¹ In addition to abnormalities of the retina (diabetic retinopathy) and the lens (cataract), various types of ocular mucosal surface disorders are also relatively common in DM patients.² They include impaired corneal sensation,^3–6 reduced tear secretion,^7,8 conjunctival squamous metaplasia and goblet cell loss,⁹ and corneal keratopathy.^2,3 Diabetic keratopathy occurs in more than 70% of diabetic patients^2–4 and increases the susceptibility of the cornea to trauma with epithelial erosions and ulcerations.^5,6,10 Although the cornea is an avascular tissue, it is the most densely innervated part of the human body, containing Aδ and unmyelinated C fibers, and derives its innervation from the ophthalmic division of the trigeminal nerve.¹¹ The sensory nerve fibers in diabetic patients with peripheral neuropathy probably undergo the earliest damage in diabetes.^12,13 Therefore, the aforementioned abnormalities also can be attributed to the decrease or loss of the nerve ends and fibers, and diabetic keratopathy also can be thought of as a form of neuropathy.^11–13 As a matter of fact, because corneal nerve structure and function can be assessed readily and accurately using in vivo corneal confocal microscopy and noncontact corneal esthesiometry, respectively, assessing corneal neuropathy has been proposed as a noninvasive and reliable way to diagnose peripheral diabetic neuropathy (keratopathy), a debilitating condition that affects approximately 50% of diabetic patients.¹⁴The cornea is a transparent tissue consisting of three cellular layers: the epithelium, stroma, and a simple epithelial layer, termed endothelium. Many major hyperglycemia-caused pathologic changes in the cornea occur around the stratified epithelia, including alterations in the epithelial basement membrane such as thickening,^6,15 a decreased number of hemidesmosomes,¹⁶ and the deposition of advanced glycation end products.^6,17 Hyperglycemia also directly affects epithelial cells and significantly alters its structure and function, resulting in basal cell degeneration,^10,18,19 decreased^20,21 or increased²² cell proliferation, superficial punctate keratitis,²³ breakdown of barrier function,^24,25 and fragility,²⁶ depending on the duration of DM and on the serum concentration of glycated hemoglobin HbA_1c. Hence, diabetic keratopathy also was termed “diabetic corneal epitheliopathy.”^27,28 Clinically, the cornea appears normal in patients with diabetic keratopathy in the absence of corneal injury. However, trauma and ocular surgeries, such as vitrectomy for vitreous hemorrhage, may require the removal of epithelial cells or damage the fragile structure, causing cell injury and/or removal of corneal epithelium. In diabetic patients, there is a considerable delay in corneal re-epithelialization after injury. The impairment of corneal epithelial wound healing could result in several types of epithelial disorders, such as persistent epithelial defects and recurrent erosion in patients after surgery.^2,29–32 Furthermore, delayed healing of the epithelial defect may be associated with sight-threatening complications, such as stromal opacification, surface irregularity, and microbial keratitis.²⁹ Hence, a better understanding of the mechanisms underlying delayed epithelial wound healing in diabetic corneas should lead to better management of the disease.We previously used a streptozocin (STZ)-induced rat model of type 1 diabetes and showed that delayed wound healing in diabetic rats is associated with the impairment of epidermal growth factor receptor–mediated cell signaling in response to mechanical injury.^21,25,28 These STZ rats had stronger rose bengal staining, decreased tear secretion, slightly attenuated sensitivity, and reduced nerve fibers. To better understand diabetic keratopathy in type 2 DM (T2D), which develops as a result of a failure to increase β-cell function and mass adequately to meet the demands of prevailing insulin resistance,³³ we maintained a colony of Goto-Kakizaki (GK) rats, one of the best characterized animal models of spontaneous T2D that were derived from Wistar rats.^34,35 We noticed that, unlike Sprague-Dawley (SD) rats, Wistar rats were less sensitive to aesthesiometer with thread and had reduced corneal sensitivity. In this study, we characterized the ocular alterations of GK rats. Our study revealed that, in addition to changes in nerve fibers, the subbasal nerve ends also were decreased. This decrease in corneal innervation may contribute to tear deficiency and delayed wound healing in GK rats.     

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.     

Targeting Mitochondria-Derived Reactive Oxygen Species to Reduce Epithelial Barrier Dysfunction and Colitis

Epithelial permeability is often increased in inflammatory bowel diseases. We hypothesized that perturbed mitochondrial function would cause barrier dysfunction and hence epithelial mitochondria could be targeted to treat intestinal inflammation. Mitochondrial dysfunction was induced in human colon-derived epithelial cell lines or colonic biopsy specimens using dinitrophenol, and barrier function was assessed by transepithelial flux of Escherichia coli with or without mitochondria-targeted antioxidant (MTA) cotreatment. The impact of mitochondria-targeted antioxidants on gut permeability and dextran sodium sulfate (DSS)–induced colitis in mice was tested. Mitochondrial superoxide evoked by dinitrophenol elicited significant internalization and translocation of E. coli across epithelia and control colonic biopsy specimens, which was more striking in Crohn’s disease biopsy specimens; the mitochondria-targeted antioxidant, MitoTEMPO, inhibited these barrier defects. Increased gut permeability and reduced epithelial mitochondrial voltage-dependent anion channel expression were observed 3 days after DSS. These changes and the severity of DSS-colitis were reduced by MitoTEMPO treatment. In vitro DSS-stimulated IL-8 production by epithelia was reduced by MitoTEMPO. Metabolic stress evokes significant penetration of commensal bacteria across the epithelium, which is mediated by mitochondria-derived superoxide acting as a signaling, not a cytotoxic, molecule. MitoTEMPO inhibited this barrier dysfunction and suppressed colitis in DSS-colitis, likely via enhancing barrier function and inhibiting proinflammatory cytokine production. These novel findings support consideration of MTAs in the maintenance of epithelial barrier function and the management of inflammatory bowel diseases.The mammalian gut harbors an immense and diverse microbiota, and host-bacteria interactions are key determinants of digestive health and general well-being.^1,2 While providing distinct benefits to the host (eg, vitamin synthesis), commensal bacteria that cross the epithelium and enter the mucosa have the potential to provoke inflammation, and movement into the circulation can result in sepsis and death. Consequently, the barrier function of the epithelium, both intrinsic (eg, epithelial tight junctions and cell membranes) and extrinsic (eg, mucus) elements, is a critical component of innate defense. Thus, the commensal bacteria-host interaction pivots on the gut epithelium as a point of first contact and knowledge of the dynamic nature of this interface is important to understanding normal intestinal function and disease.³The epithelial barrier is not static; rather, it is highly dynamic and tightly regulated to maintain gut homeostasis. However, uncontrolled or prolonged increases in gut permeability have the potential to initiate or exaggerate enteric inflammatory disease. For example, the consensus on the etiology of inflammatory bowel disease (IBD; Crohn’s disease or ulcerative colitis) is that disease develops because of an inappropriate immune response to the gut microbiota in a genetically susceptible individual.⁴ This suggests a barrier defect because microbes, or their products, in the gut lumen must access the mucosal immune system by crossing the epithelial cell layer. Although increased epithelial permeability as a primary cause of IBD remains unproved,³ the leaky gut hypothesis⁵ is supported by observations in experimental models of colitis^3,6,7 and increased epithelial permeability has been repeatedly demonstrated in active IBD.^8,9 Therefore, the ability to enhance the barrier property of the epithelium would be of value in ameliorating enteric inflammatory disease.Given that control of epithelial permeability (apical junction complex formation and transcellular permeation) is energy dependent, mitochondria should be essential for appropriate regulation of barrier function. Indeed, factors such as infection, nonsteroidal anti-inflammatory drugs, and smoking, which can contribute to the pathophysiological characteristics of Crohn’s disease, also perturb mitochondrial function.^10–12 Furthermore, structurally abnormal mitochondria have been observed in tissue from patients with gut inflammation,¹³ in animal models of gut disease,¹⁴ and in epithelial monolayers treated with bacterial toxins or low-grade pathogens.¹⁵ However, despite these findings and the keen interest in mitochondria in the pathophysiological characteristics of neuromuscular disease, diabetes, and obesity,^16–18 there are limited data on the role of mitochondria in colitis.We have previously shown that model epithelia treated with dinitrophenol (DNP) to uncouple oxidative phosphorylation display decreased barrier function, as characterized by lower transepithelial resistance (TER) indicative of increased paracellular permeability, and the internalization and translocation of noninvasive, nontoxigenic commensal E. coli.^13,19 The latter is particularly intriguing for two reasons: entry of commensal bacteria into the enterocyte likely represents a threat and the enterocytes’ response (eg, IL-8 synthesis) could promote inflammation; and the contribution of transcellular permeability to a barrier defect is not well understood and needs to be comprehensively assessed and fully integrated to any consideration of the epithelial barrier and innate defense. Thus, the current study was designed to uncover the mechanism(s) underlying the increased internalization and translocation of commensal Escherichia coli across gut epithelia, which occurs as a consequence of metabolic stress evoked by targeted perturbation of mitochondrial function.     

A Tissue-Specific Role for Nlrp3 in Tubular Epithelial Repair after Renal Ischemia/Reperfusion

Ischemia/reperfusion injury is a major cause of acute kidney injury. Improving renal repair would represent a therapeutic strategy to prevent renal dysfunction. The innate immune receptor Nlrp3 is involved in tissue injury, inflammation, and fibrosis; however, its role in repair after ischemia/reperfusion is unknown. We address the role of Nlrp3 in the repair phase of renal ischemia/reperfusion and investigate the relative contribution of leukocyte- versus renal-associated Nlrp3 by studying bone marrow chimeric mice. We found that Nlrp3 expression was most profound during the repair phase. Although Nlrp3 expression was primarily expressed by leukocytes, both leukocyte- and renal-associated Nlrp3 was detrimental to renal function after ischemia/reperfusion. The Nlrp3-dependent cytokine IL-1β remained unchanged in kidneys of all mice. Leukocyte-associated Nlrp3 negatively affected tubular apoptosis in mice that lacked Nlrp3 expression on leukocytes, which correlated with reduced macrophage influx. Nlrp3-deficient (Nlrp3KO) mice with wild-type bone marrow showed an improved repair response, as seen by a profound increase in proliferating tubular epithelium, which coincided with increased hepatocyte growth factor expression. In addition, Nlrp3KO tubular epithelial cells had an increased repair response in vitro, as seen by an increased ability of an epithelial monolayer to restore its structural integrity. In conclusion, Nlrp3 shows a tissue-specific role in which leukocyte-associated Nlrp3 is associated with tubular apoptosis, whereas renal-associated Nlrp3 impaired wound healing.Ischemia/reperfusion (IR) injury is a major cause of acute kidney injury¹ and increases the risk of developing chronic kidney disease (CKD).² After injury, wounded tissue organizes an efficient response that aims to combat infections, clear cell debris, re-establish cell number, and reorganize tissue architecture. First, necrotic tissue releases danger-associated molecular patterns, such as high-mobility group box-1³ or mitochondrial DNA,⁴ which leads to chemokine secretion⁵ and a subsequent influx of leukocytes. Second, neutrophils and macrophages clear cellular debris but also increase renal damage because depletion of neutrophils⁶ or macrophages within 48 hours of IR will reduce renal damage.⁷ At approximately 72 hours of reperfusion, the inflammatory phase transforms into the repair phase and is characterized by surviving tubular epithelial cells (TECs) that dedifferentiate, migrate, and proliferate to restore renal function.⁸Previously, we have shown that Toll-like receptor (TLR) 2 and TLR4 play a detrimental role after acute renal IR injury.^{9, 10, 11} In addition, TLR2 appeared also pivotal in mediating tubular repair in vitro after cisplatin-induced injury,¹² indicating a dual role for TLR2. The cytosolic innate immune receptor Nlrp3 is able to sense cellular damage¹³ and mediates renal inflammation and pathological characteristics after IR^{14, 15, 16} or nephrocalcinosis.¹⁷ Next to the detrimental role of Nlrp3 in different renal disease models and consistent with the dual role of TLR2, Nlrp3 was shown to protect against loss of colonic epithelial integrity.¹⁸ We, therefore, speculate that Nlrp3, which contributes to sterile renal inflammation during acute renal IR injury, might also drive subsequent tubular repair.To test this hypothesis, we investigated the role of leukocyte- versus renal-associated Nlrp3 with respect to tissue repair after renal IR. We observed that both renal- and leukocyte-associated Nlrp3s are detrimental to renal function after renal IR injury; however, this is through different mechanisms. Leukocyte-associated Nlrp3 is related to increased tubular epithelial apoptosis, whereas renal-associated Nlrp3 impairs the tubular epithelial repair response. Our data suggest Nlrp3 as a negative regulator of resident tubular cell proliferation in addition to its detrimental role in renal fibrosis and inflammation.^{14, 19}     

Glycosaminoglycan Regulation by VEGFA and VEGFC of the Glomerular Microvascular Endothelial Cell Glycocalyx in?Vitro

Damage to endothelial glycocalyx impairs vascular barrier function and may contribute to progression of chronic vascular disease. An early indicator is microalbuminuria resulting from glomerular filtration barrier damage. We investigated the contributions of hyaluronic acid (HA) and chondroitin sulfate (CS) to glomerular microvascular endothelial cell (GEnC) glycocalyx and examined whether these are modified by vascular endothelial growth factors A and C (VEGFA and VEGFC). HA and CS were imaged on GEnCs and their resynthesis was examined. The effect of HA and CS on transendothelial electrical resistance (TEER) and labeled albumin flux across monolayers was assessed. Effects of VEGFA and VEGFC on production and charge characteristics of glycosaminoglycan (GAG) were examined via metabolic labeling and liquid chromatography. GAG shedding was quantified using Alcian Blue. NDST2 expression was examined using real-time PCR. GEnCs expressed HA and CS in the glycocalyx. CS contributed to the barrier to both ion (TEER) and protein flux across the monolayer; HA had only a limited effect. VEGFC promoted HA synthesis and increased the charge density of synthesized GAGs. In contrast, VEGFA induced shedding of charged GAGs. CS plays a role in restriction of macromolecular flux across GEnC monolayers, and VEGFA and VEGFC differentially regulate synthesis, charge, and shedding of GAGs in GEnCs. These observations have important implications for endothelial barrier regulation in glomerular and other microvascular beds.The apical side of endothelial cells is coated with an endothelial surface layer (ESL) composed of a surface-anchored glycocalyx which is itself composed of negatively charged proteoglycans [proteins with glycosaminoglycan (GAG) side chains] and glycoproteins (glycosylated proteins) and a more loosely associated layer of adsorbed plasma proteins.¹ The endothelial glycocalyx is 200 nm to 2 μm thick (depending on the vascular bed and also on the visualization technique).²The glycocalyx mediates shear, attenuates leukocyte and platelet adhesion,² regulates systemic vascular permeability,^3–6 and allows free passage of solutes but limits passage of charged macromolecules.⁷ The ESL is damaged in reperfusion injury, inflammation and trauma, hypervolemia, atherosclerosis, and diabetes (summarized by Becker et al²). ESL thickness is reduced by hyperglycemic infusions in healthy subjects, correlating with endothelial dysfunction and an increase in vascular permeability,⁸ and in type 1 diabetes correlating with microalbuminuria,⁹ suggesting a direct link between endothelial ESL dysfunction and this dysfunction of the glomerular filtration barrier. In mice, infusions of the GAG-specific enzymes hyaluronidase, chondroitinase, and heparinase reduced the glomerular endothelial cell (GEnC) ESL thickness, resulting in reduced charge selectivity and increased macromolecular passage (proteinuria).¹⁰ In addition, proteinuria is accompanied with a loss of charge selectivity and a reduction in the core proteins decorin, fibromodulin, and versican in nonobese diabetic mice.¹¹ Salmon et al¹² demonstrated an age-related reduction in ESL in glomerular (and other) microvessels of Munich Wistar Frömter rats; this was accompanied by an increase in glomerular albumin permeability, which could be rescued by intravenous injections of lectin. Finally, using doxorubicin (Adriamycin) to induce proteinuria in mice, Jeansson et al¹³ demonstrated increased fractional clearance of larger molecules in cooled, isolated, perfused kidneys, as well as reduced charge in the glomerular filtration barrier; this was accompanied by reduced synthesis of some core proteins and GAGs in the isolated glomeruli and reduced thickness of the ESL. Taken together, these studies strongly suggest that the ESL also regulates permeability in the glomerular filtration barrier. The glomerular filtration barrier is a tightly regulated filter that restricts macromolecular protein passage while allowing filtration of water and small solutes. Dysfunction of this barrier results in proteinuria, a hallmark of kidney disease. The glomerular filtration barrier consists of a trilayer of GEnCs, a glomerular basement membrane and glomerular epithelial cells (podocytes) whose foot processes interdigitate around the glomerular microvessels. Each of these layers contributes to the regulation of glomerular macromolecular permeability.^1,14 The GEnC contribution is increasingly thought to be largely dependent on the ESL.^10,15,16We have previously studied the GEnC ESL in vitro in a human conditionally immortalized (ci) cell line and, using electron microscopy, revealed the presence of an ESL layer measuring 200 nm, which is consistent with recent sophisticated measurements of endothelial glycocalyx in vivo.¹⁷ We confirmed the presence of proteoglycan core proteins, as previously demonstrated on human GEnCs,¹⁸ and of heparan sulfate (HS), a sulfated GAG.¹⁹ Enzymatic removal of HS increased macromolecular protein passage across a monolayer by 40%, whereas electrical resistance (a measure of pathways open to water and small molecules) was not affected. Furthermore, we have shown that high-glucose conditions reduce GEnC ESL and lead to a corresponding increase in macromolecular protein passage across GEnC monolayers,²⁰ demonstrating that the GEnC glycocalyx is present in vitro and plays a functional role.Glomerular enzyme infusion studies by Jeansson et al¹⁰ implicated, in addition to removal of HS, removal of hyaluronic acid (HA) and chondroitin sulfate (CS) in increased fractional albumin clearance. HA and CS are also thought to play a role in systemic macromolecular permeability.²¹ In contrast to other GAGs, HA is not synthesized in the Golgi apparatus, but rather at the plasma membrane (by HA synthases 1 to 3), and it is not attached to core proteins (reviewed by Genasetti et al²²). Furthermore, HA is unbranched and unsulfated, and therefore it does not have a strong negative charge. CS is sulfated on assembly within the Golgi apparatus, where it becomes attached to one of its core proteins (eg, aggrecan or versican), forming a proteoglycan. In the present study, we aimed to determine the contribution of HA and CS to the GEnC glycocalyx through in vitro studies using unique human ciGEnCs and selective enzymes.Vascular endothelial growth factor A (VEGFA), originally called vascular permeability factor, is a powerful angiogenic growth factor that has profound effects on vascular endothelial behavior (as summarized by Tammela et al²³), including GEnC maintenance,²⁴ repair,²⁵ and permeability.²⁶ VEGFA is highly expressed by podocytes.²⁷ Another member of the VEGF family of proteins, VEGFC, is a lymphangiogenic growth factor that can stimulate similar pathways to those of VEGFA in both lymphatic and vascular endothelial cells.²⁸ VEGFC also is expressed by podocytes.²⁹ Podocyte–endothelial signaling through VEGF is vital for GEnC maintenance and permeability regulation. It has been postulated that the effects of VEGFA on microvessel permeability are due in part to partial degradation of the glycocalyx.³⁰ In the present study, we aimed to determine whether VEGFs can modify the GEnC glycocalyx. Our central hypothesis was that CS and HA contribute to GEnC barrier maintenance and that they are modified by VEGFs.     

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.     

Rac1 and Cdc42 Differentially Modulate Cigarette Smoke–Induced Airway Cell Migration through p120-Catenin–Dependent and –Independent Pathways

The adherens junction protein p120-catenin (p120ctn) shuttles between E-cadherin–bound and cytoplasmic pools to regulate E-cadherin/catenin complex stability and cell migration, respectively. When released from the adherens junction, p120ctn promotes cell migration through modulation of the Rho GTPases Rac1, Cdc42, and RhoA. Accordingly, the down-regulation and cytoplasmic mislocalization of p120ctn has been reported in all subtypes of lung cancers and is associated with grave prognosis. Previously, we reported that cigarette smoke induced cytoplasmic translocation of p120ctn and cell migration, but the underlying mechanism was unclear. Using primary human bronchial epithelial cells exposed to smoke-concentrated medium (Smk), we observed the translocation of Rac1 and Cdc42, but not RhoA, to the leading edge of polarized and migrating human bronchial epithelial cells. Rac1 and Cdc42 were robustly activated by smoke, whereas RhoA was inhibited. Accordingly, siRNA knockdown of Rac1 or Cdc42 completely abolished Smk-induced cell migration, whereas knockdown of RhoA had no effect. p120ctn/Rac1 double knockdown completely abolished Smk-induced cell migration, whereas p120ctn/Cdc42 double knockdown did not. These data suggested that Rac1 and Cdc42 coactivation was essential to smoke-promoted cell migration in the presence of p120ctn, whereas migration proceeded via Rac1 alone in the absence of p120ctn. Thus, Rac1 may provide an omnipotent therapeutic target in reversing cell migration during the early (intact p120ctn) and late (loss of p120ctn) stages of lung carcinogenesis.Cigarette smoke contains >4000 active constituents, ≥60 of which are established carcinogens and/or mutagens.¹ With a 20-fold greater risk of lung cancer and accounting for 87% of lung cancer–related deaths,² smoking continues to represent the single most important carcinogenic exposure. Because treatment of lung cancer is largely ineffective, recent research has been focused on efforts to identify and reverse early events leading to the initiation of lung cancer by smoke.³ Emerging evidence suggests that smoke mediates epithelial-mesenchymal transition (EMT) and pretumor cell migration by disrupting cell-cell adhesion in polarized mucosal epithelia.^{4, 5} During EMT, cells switch from a polarized immobile epithelial phenotype to a highly motile fibroblast phenotype.⁶ Unregulated EMT confers epithelial cells with stem cell–like properties capable of self-renewal, metastasis, and resistance to apoptosis.^{6, 7} Little is known about how smoke mediates EMT during the early stages of lung cancer.E-cadherin (E-cad)–based adherens junctions (AJs) interact with catenins to modulate cell-cell adhesion.⁸ Structural analysis by X-ray crystallography revealed that p120-catenin (p120ctn) binds to the juxtamembrane domain of E-cad, where it regulates stability and turnover of E-cad by concealing the juxtamembrane domain residues implicated in endocytosis and ubiquitination of E-cad.^{9, 10} The disruption of p120ctn leads to E-cad degradation, a major hallmark of EMT and malignancy.⁸ Accumulating evidence suggests that p120ctn shuttles between E-cad–bound and cytoplasmic pools. When bound to E-cad, p120ctn stabilizes the AJ and acts as a tumor and/or metastasis suppressor.¹¹ When released from the AJ, p120ctn can promote EMT and cell migration through the degradation of E-cad and the modulation of Rho GTPase activity, respectively.^{8, 11, 12, 13, 14, 15, 16, 17} Accordingly, membrane loss, down-regulation, and cytoplasmic mislocalization of E-cad and p120ctn have been reported in most epithelial cancers, including all subtypes of lung cancers, and are frequently associated with a grave prognosis.^{18, 19}In lung cancer, ectopic cytoplasmic expression of p120ctn and E-cad has been associated with elevated expression of Rho GTPases.¹⁹ Rac1, Cdc42, and RhoA shuttle between their inactive GDP– and active GTP–bound forms to regulate the dynamics of the actin cytoskeleton, cell motility, cadherin-dependent adhesion, and cell proliferation.^{20, 21, 22} Lamellipodia, filopodia, and stress fibers are regarded as typical phenotypes of activated Rac1, Cdc42, and RhoA, respectively.²³ Active Rac1 and Cdc42 drive protrusion formation at the leading edge of a migrating leukocyte, whereas active RhoA aggregates at the rear and sides of the cell, preventing protrusion formation.²¹ p120ctn can act as a guanine nucleotide dissociation inhibitor to inhibit RhoA through preferential interaction and sequestration of RhoA in its GDP-bound form.¹² Alternatively, p120ctn indirectly activates Rac1 and Cdc42 through its interaction with Vav2, a guanine nucleotide exchange factor that promotes the exchange of GDP with GTP.^{13, 14}We sought to investigate the role of p120ctn in regulating Rho GTPase activity in the initiating stages of cigarette smoke–induced cell migration. Given the opposing roles of membrane versus cytoplasmic p120ctn in carcinogenesis, this study was performed in primary human bronchial epithelial (HBE) cells with intact AJs. Realizing that cancer is a multistep process requiring numerous chemically mediated insults, we mimicked the exposure of airway epithelial cells to smoke using an established model of smoke-conditioned medium (Smk).^{24, 25} Primary HBE cells exposed to Smk medium underwent malignant transformation in 8 days, demonstrating rapid proliferation, anchorage-independent growth, and tumorigenesis in nude mice.²⁶ With this approach, we discovered p120ctn-dependent and p120ctn-independent pathways mediating cell migration provoked by cigarette smoke. In the presence of p120ctn, coactivation of Rac1 and Cdc42 was essential to promote cell migration, whereas in the absence of p120ctn, activation of Rac1 alone induced migration. These data reveal new details regarding the molecular events promoting cell migration in the earliest stages of cigarette smoke–induced tumorigenesis and open the way for novel approaches to the prevention of lung cancer in smokers.