Aiping Zhao

Identification of human zonulin, a physiological modulator of tight junctions, as prehaptoglobin-2

Increased intestinal permeability (IP) has emerged recently as a common underlying mechanism in the pathogenesis of allergic, inflammatory, and autoimmune diseases. The characterization of zonulin, the only physiological mediator known to regulate IP reversibly, has remained elusive. Through proteomic analysis of human sera, we have now identified human zonulin as the precursor for haptoglobin-2 (pre-HP2). Although mature HP is known to scavenge free hemoglobin (Hb) to inhibit its oxidative activity, no function has ever been ascribed to its uncleaved precursor form. We found that the single-chain zonulin contains an EGF-like motif that leads to transactivation of EGF receptor (EGFR) via proteinase-activated receptor 2 (PAR2) activation. Activation of these 2 receptors was coupled to increased IP. The siRNA-induced silencing of PAR2 or the use of PAR2−/− mice prevented loss of barrier integrity. Proteolytic cleavage of zonulin into its α2- and β-subunits neutralized its ability to both activate EGFR and increase IP. Quantitative gene expression revealed that zonulin is overexpressed in the intestinal mucosa of subjects with celiac disease. To our knowledge, this is the initial example of a molecule that exerts a biological activity in its precursor form that is distinct from the function of its mature form. Our results therefore characterize zonulin as a previously undescribed ligand that engages a key signalosome involved in the pathogenesis of human immune-mediated diseases that can be targeted for therapeutic interventions.

Th2 Cytokine-Induced Alterations in Intestinal Smooth Muscle Function Depend on Alternatively Activated Macrophages

BACKGROUND & AIMS Enteric nematode infection induces a strong type 2 T helper cell (Th2) cytokine response characterized by increased infiltration of various immune cells, including macrophages. The role of these immune cells in host defense against nematode infection remains poorly defined. The present study investigated the role of macrophages and the arginase pathway in nematode-induced changes in intestinal smooth muscle function and worm expulsion. METHODS Mice were infected with Nippostrongylus brasiliensis and treated with clodronate-containing liposome to deplete macrophages or given S-(2-boronoethyl)-I-cysteine in drinking water to inhibit arginase activity. Segments of intestinal smooth muscle were suspended in organ baths to determine responses to acetylcholine, 5-hydroxytryptamine, or nerve stimulation. The phenotype of macrophages was monitored by measuring mRNA expression of the specific molecular markers by real-time polymerase chain reaction or viewed by immunofluorescence staining. RESULTS Infection increased the infiltration of macrophages and up-regulation alternatively activated macrophage markers by a mechanism dependent on interleukin-4 (IL-4) or interleukin-13 (IL-13) activation of signal transducer and activator of transcription 6. Elimination of alternatively activated macrophages blocked smooth muscle hypercontractility and the increased smooth muscle thickness, and impaired worm expulsion. In addition, specific inhibition of arginase activity interfered with smooth muscle contractility, but only partially affected the protective immunity of the host. CONCLUSIONS These data show that the phenotype of macrophages is determined by the local immune environment and that alternatively activated macrophages play a major role in the effects of Th2 cytokines, IL-4 and IL-13, on intestinal smooth muscle function.

Localized Multigene Expression Patterns Support an Evolving Th1/Th2-Like Paradigm in Response to Infections with Toxoplasma gondii and Ascaris suum

ABSTRACT Human infectious diseases have been studied in pigs because the two species have common microbial, parasitic, and zoonotic organisms, but there has been no systematic evaluation of cytokine gene expression in response to infectious agents in porcine species. In this study, pigs were inoculated with two clinically and economically important parasites, Toxoplasma gondii and Ascaris suum, and gene expression in 11 different tissues for 20 different swine Th1/Th2-related cytokines, cytokine receptors, and markers of immune activation were evaluated by real-time PCR. A generalized Th1-like pattern of gene expression was evident in pigs infected with T. gondii, along with an increased anti-inflammatory gene expression pattern during the recovery phase of the infection. In contrast, an elevated Th2-like pattern was expressed during the period of expulsion of A. suum fourth-stage larvae from the small intestine of pigs, along with low-level Th1-like and anti-inflammatory cytokine gene expression. Prototypical immune and physiological markers of infection were observed in bronchial alveolar lavage cells, small intestinal smooth muscle, and epithelial cells. This study validated the use of a robust quantitative gene expression assay to detect immune and inflammatory markers at multiple host tissue sites, enhanced the definition of two important swine diseases, and supported the use of swine as an experimental model for the study of immunity to infectious agents relevant to humans.

Membrane-anchored serine protease matriptase regulates epithelial barrier formation and permeability in the intestine

The intestinal epithelium serves as a major protective barrier between the mammalian host and the external environment. Here we show that the transmembrane serine protease matriptase plays a pivotol role in the formation and integrity of the intestinal epithelial barrier. St14 hypomorphic mice, which have a 100-fold reduction in intestinal matriptase mRNA levels, display a 35% reduction in intestinal transepithelial electrical resistance (TEER). Matriptase is expressed during intestinal epithelial differentiation and colocalizes with E-cadherin to apical junctional complexes (AJC) in differentiated polarized Caco-2 monolayers. Inhibition of matriptase activity using a specific peptide inhibitor or by knockdown of matriptase by siRNA disrupts the development of TEER in barrier-forming Caco-2 monolayers and increases paracellular permeability to macromolecular FITC-dextran. Loss of matriptase was associated with enhanced expression and incorporation of the permeability-associated, “leaky” tight junction protein claudin-2 at intercellular junctions. Knockdown of claudin-2 enhanced the development of TEER in matriptase-silenced Caco-2 monolayers, suggesting that the reduced barrier integrity was caused, at least in part, by an inability to regulate claudin-2 expression and incorporation into junctions. We find that matriptase enhances the rate of claudin-2 protein turnover, and that this is mediated indirectly through an atypical PKCζ-dependent signaling pathway. These results support a key role for matriptase in regulating intestinal epithelial barrier competence, and suggest an intriguing link between pericellular serine protease activity and tight junction assembly in polarized epithelia.

Critical Role of IL-25 in Nematode Infection-Induced Alterations in Intestinal Function

IL-25 (IL-17E) is a member of the IL-17 cytokine family. IL-25–deficient mice exhibit impaired Th2 immunity against nematode infection, implicating IL-25 as a key component in mucosal immunity. The sources of IL-25 and mechanisms responsible for the induction of Th2 immunity by IL-25 in the gastrointestinal tract remain poorly understood. There is also little information on the regulation of IL-25 during inflammation or its role in gut function. In the current study, we investigated the regulation of IL-25 during Nippostrongylus brasiliensis infection and the contribution of IL-25 to the infection-induced alterations in intestinal function. We found that epithelial cells, but not immune cells, are the major source of IL-25 in the small intestine. N. brasiliensis infection-induced upregulation of IL-25 depends upon IL-13 activation of STAT6. IL-25−/− mice had diminished intestinal smooth muscle and epithelial responses to N. brasiliensis infection that were associated with an impaired Th2 protective immunity. Exogenous IL-25 induced characteristic changes similar to those after nematode infection but was unable to restore the impaired host immunity against N. brasiliensis infection in IL-13−/− mice. These data show that IL-25 plays a critical role in nematode infection-induced alterations in intestinal function that are important for host protective immunity, and IL-13 is the major downstream Th2 cytokine responsible for the IL-25 effects.

Macrophages as IL-25/IL-33-Responsive Cells Play an Important Role in the Induction of Type 2 Immunity

Type 2 immunity is essential for host protection against nematode infection but is detrimental in allergic inflammation or asthma. There is a major research focus on the effector molecules and specific cell types involved in the initiation of type 2 immunity. Recent work has implicated an important role of epithelial-derived cytokines, IL-25 and IL-33, acting on innate immune cells that are believed to be the initial sources of type 2 cytokines IL-4/IL-5/IL-13. The identities of the cell types that mediate the effects of IL-25/IL-33, however, remain to be fully elucidated. In the present study, we demonstrate that macrophages as IL-25/IL-33-responsive cells play an important role in inducing type 2 immunity using both in vitro and in vivo approaches. Macrophages produced type 2 cytokines IL-5 and IL-13 in response to the stimulation of IL-25/IL-33 in vitro, or were the IL-13-producing cells in mice administrated with exogenous IL-33 or infected with Heligmosomoides bakeri. In addition, IL-33 induced alternative activation of macrophages primarily through autocrine IL-13 activating the IL-4Rα-STAT6 pathway. Moreover, depletion of macrophages attenuated the IL-25/IL-33-induced type 2 immunity in mice, while adoptive transfer of IL-33-activated macrophages into mice with a chronic Heligmosomoides bakeri infection induced worm expulsion accompanied by a potent type 2 protective immune response. Thus, macrophages represent a unique population of the innate immune cells pivotal to type 2 immunity and a potential therapeutic target in controlling type 2 immunity-mediated inflammatory pathologies.

Anti-Inflammatory Mechanisms of Enteric Heligmosomoides polygyrus Infection against Trinitrobenzene Sulfonic Acid-Induced Colitis in a Murine Model

ABSTRACT Recent studies showed that enteric helminth infection improved symptoms in patients with inflammatory bowel disease as well as in experimental models of colitis. The aim of this study was to determine the mechanism of the protective effect of helminth infection on colitis-induced changes in immune and epithelial cell function. BALB/c mice received an oral infection of Heligmosomoides polygyrus third-stage larvae, were given intrarectal saline or trinitrobenzene sulfonic acid (TNBS) on day 10 postinfection, and were studied 4 days later. Separate groups of mice received intrarectal saline or TNBS on day 10 and were studied on day 14. Muscle-free colonic mucosae were mounted in Ussing chambers to measure mucosal permeability and secretion. Expression of cytokines was assessed by quantitative real-time PCR, and mast cells were visualized by immunohistochemistry. TNBS-induced colitis induced mucosal damage, upregulated Th1 cytokines, and depressed secretory responses. Heligmosomoides polygyrus elevated Th2 cytokine expression, increased mast cell infiltration and mucosal resistance, and also reduced some secretory responses. Prior H. polygyrus infection prevented TNBS-induced upregulation of Th1 cytokines and normalized secretory responses to specific agonists. TNBS-induced colitis did not alter H. polygyrus-induced mast cell infiltration or upregulation of Th2 cytokine expression. The results indicate that the protective mechanism of enteric nematode infection against TNBS-induced colitis involves prevention of Th1 cytokine expression and improved colonic function by a mechanism that may involve mast cell-mediated protection of neural control of secretory function. Similar response patterns could account for the clinical improvement seen in inflammatory bowel disease with helminthic therapy.

Mast Cell-Mediated Changes in Smooth Muscle Contractility during Mouse Giardiasis

ABSTRACT Giardia intestinalis is a significant cause of diarrheal disease worldwide. Infections in animal models have been shown to cause changes in gastrointestinal transit that depend on adaptive immune responses and are mediated, in part, through neuronal nitric oxide synthase. Nitric oxide is an inhibitory neurotransmitter, and we therefore investigated potential excitatory pathways that might be involved in the response to Giardia infection. Infected mice exhibited increased spontaneous and cholecystokinin (CCK)-induced contractions of longitudinal smooth muscle. In contrast, enhanced contractile responses were not observed in response to acetylcholine, 5-hydroxytryptamine, or the protease-activated receptor-1 agonist peptide TFFLR. Giardia-induced changes in smooth muscle function appear to be mediated primarily by mast cells, as both spontaneous and CCK-induced contractions were blocked by pretreatment with either ketotifen or compound 48/80. Together, these data support a model in which CCK release triggers mast cell degranulation, leading to increases in smooth muscle contractility. These contractions, coupled with nitric oxide-mediated muscle relaxation, promote intestinal transit and parasite elimination.

Parasitic Nematode-Induced Modulation of Body Weight and Associated Metabolic Dysfunction in Mouse Models of Obesity

ABSTRACT Obesity is associated with a chronic low-grade inflammation characterized by increased levels of proinflammatory cytokines that are implicated in disrupted metabolic homeostasis. Parasitic nematode infection induces a polarized Th2 cytokine response and has been explored to treat autoimmune diseases. We investigated the effects of nematode infection against obesity and the associated metabolic dysfunction. Infection of RIP2-Opa1KO mice or C57BL/6 mice fed a high-fat diet (HFD) with Nippostrongylus brasiliensis decreased weight gain and was associated with improved glucose metabolism. Infection of obese mice fed the HFD reduced body weight and adipose tissue mass, ameliorated hepatic steatosis associated with a decreased expression of key lipogenic enzymes/mediators, and improved glucose metabolism, accompanied by changes in the profile of metabolic hormones. The infection resulted in a phenotypic change in adipose tissue macrophages that was characterized by upregulation of alternative activation markers. Interleukin-13 (IL-13) activation of the STAT6 signaling pathway was required for the infection-induced attenuation of steatosis but not for improved glucose metabolism, whereas weight loss was attributed to both IL-13/STAT6-dependent and -independent mechanisms. Parasitic nematode infection has both preventive and therapeutic effects against the development of obesity and associated features of metabolic dysfunction in mice.

Dissociation of Endotoxin Tolerance and Differentiation of Alternatively Activated Macrophages

Endotoxin tolerance is a complex phenomenon characterized primarily by decreased production of proinflammatory cytokines, chemokines, and other inflammatory mediators, whereas the expression of other genes are induced or unchanged. Endotoxin tolerance is induced by prior exposure of murine macrophages/human monocytes, experimental animals, or people to TLR ligands. Although recent studies reported a possible relationship between endotoxin tolerance and differentiation of alternatively activated macrophages (AA-MΦs or M2), we show in this study that LPS pretreatment of IL-4Rα−/− and STAT6−/− macrophages, which fail to develop into AA-MΦs, resulted in tolerance of proinflammatory cytokines, as well as molecules and chemokines previously associated with AA-MΦs (e.g., arginase-1, mannose receptor, CCL2, CCL17, and CCL22). In contrast to LPS, wild-type (WT) MΦs pretreated with IL-4, the prototype inducer of AA-MΦs, did not induce endotoxin tolerance with respect to proinflammatory cytokines, AA-MΦ–associated chemokines, negative regulators, NF-κB binding and subunit composition, and MAPKs; conversely, IL-13−/− macrophages were tolerized equivalently to WT MΦs by LPS pretreatment. Further, IL-4Rα deficiency did not affect the reversal of endotoxin tolerance exerted by the histone deacetylase inhibitor trichostatin A. Like WT mice, 100% of LPS-tolerized IL-4Rα–deficient mice survived LPS + d-galactosamine–induced lethal toxicity and exhibited decreased serum levels of proinflammatory cytokines and AA-MΦ–associated chemokines induced by LPS challenge compared with nontolerized mice. These data indicate that the signaling pathways leading to endotoxin tolerance and differentiation of AA-MΦs are dissociable.