Targeting PP2A and proteasome activity ameliorates features of allergic airway disease in mice.

BACKGROUND: Asthma is an allergic airway disease (AAD) caused by aberrant immune responses to allergens. Protein phosphatase-2A (PP2A) is an abundant serine/threonine phosphatase with anti-inflammatory activity. The ubiquitin proteasome system (UPS) controls many cellular processes, including the initiation of inflammatory responses by protein degradation. We assessed whether enhancing PP2A activity with fingolimod (FTY720) or 2-amino-4-(4-(heptyloxy) phenyl)-2-methylbutan-1-ol (AAL(S) ), or inhibiting proteasome activity with bortezomib (BORT), could suppress experimental AAD. METHODS: Acute AAD was induced in C57BL/6 mice by intraperitoneal sensitization with ovalbumin (OVA) in combination with intranasal (i.n) exposure to OVA. Chronic AAD was induced in mice with prolonged i.n exposure to crude house dust mite (HDM) extract. Mice were treated with vehicle, FTY720, AAL(S) , BORT or AAL(S) +BORT and hallmark features of AAD assessed. RESULTS: AAL(S) reduced the severity of acute AAD by suppressing tissue eosinophils and inflammation, mucus-secreting cell (MSC) numbers, type 2-associated cytokines (interleukin (IL)-33, thymic stromal lymphopoietin, IL-5 and IL-13), serum immunoglobulin (Ig)E and airway hyper-responsiveness (AHR). FTY720 only suppressed tissue inflammation and IgE. BORT reduced bronchoalveolar lavage fluid (BALF) and tissue eosinophils and inflammation, IL-5, IL-13 and AHR. Combined treatment with AAL(S) +BORT had complementary effects and suppressed BALF and tissue eosinophils and inflammation, MSC numbers, reduced the production of type 2 cytokines and AHR. AAL(S) , BORT and AAL(S) +BORT also reduced airway remodelling in chronic AAD. CONCLUSION: These findings highlight the potential of combination therapies that enhance PP2A and inhibit proteasome activity as novel therapeutic strategies for asthma.

Effect of neonatal respiratory infection on adult BALB/c hippocampal glucocorticoid and mineralocorticoid receptors.

The current study investigated the effects of neonatal infection with Chlamydia muridarum bacteria on glucocorticoid (GR) and mineralocorticoid (MR) receptors in the adult mouse hippocampus. In male adults infected at birth, circulating corticosterone was significantly increased when compared to same sex controls; while neonatal infection resulted in female adults with significantly increased GR mRNA compared to same sex controls. When comparing males and females after neonatal infection, males had significantly less GR protein than females. Interestingly, after control treatment, males had significantly more GR mRNA, MR mRNA, and GR protein with significantly lower corticosterone than females. Neonatal respiratory infection significantly impacts adult hippocampal GR and MR, and circulating corticosterone in a sex-specific manner potentially altering stress responsivity.

Streptococcus pneumoniae infection suppresses allergic airways disease by inducing regulatory T-cells.

An inverse association exists between some bacterial infections and the prevalence of asthma. We investigated whether Streptococcus pneumoniae infection protects against asthma using mouse models of ovalbumin (OVA)-induced allergic airway disease (AAD). Mice were intratracheally infected or treated with killed S. pneumoniae before, during or after OVA sensitisation and subsequent challenge. The effects of S. pneumoniae on AAD were assessed. Infection or treatment with killed S. pneumoniae suppressed hallmark features of AAD, including antigen-specific T-helper cell (Th) type 2 cytokine and antibody responses, peripheral and pulmonary eosinophil accumulation, goblet cell hyperplasia, and airway hyperresponsiveness. The effect of infection on the development of specific features of AAD depended on the timing of infection relative to allergic sensitisation and challenge. Infection induced significant increases in regulatory T-cell (Treg) numbers in lymph nodes, which correlated with the degree of suppression of AAD. Tregs reduced T-cell proliferation and Th2 cytokine release. The suppressive effects of infection were reversed by anti-CD25 treatment. Respiratory infection or treatment with S. pneumoniae attenuates allergic immune responses and suppresses AAD. These effects may be mediated by S. pneumoniae-induced Tregs. This identifies the potential for the development of therapeutic agents for asthma from S. pneumoniae.

Neonatal respiratory infection and adult re-infection: effect on glucocorticoid and mineralocorticoid receptors in the hippocampus in BALB/c mice.

Stressful events during the perinatal period in both humans and animals have long-term consequences for the development and function of physiological systems and susceptibility to disease in adulthood. One form of stress commonly experienced in the neonatal period is exposure to bacterial and viral infections. The current study investigated the effects of live Chlamydia muridarum bacterial infection at birth followed by re-infection in adulthood on hippocampal glucocorticoid receptors (GR) and mineralocorticoid receptors (MR) and stress response outcomes. Within 24 h of birth, neonatal mice were infected intranasally with C. muridarum (400 inclusion-forming units [ifu]) or vehicle. At 42 days, mice were re-infected (100 ifu) and euthanized 10 days later. In males, infection in adulthood alone had the most impact on the parameters measured with significant increases in GR protein compared to adult infection alone; and significant increases MR protein and circulating corticosterone compared to other treatment groups. Neonatal infection alone induced the largest alterations in the females with results showing reciprocal patterns for GR protein and TH protein. Perinatal infection resulted in a blunted response following adult infection for both males and females across all parameters. The present study demonstrates for the first time that males and females respond differently to infection based on the timing of the initial insult and that there is considerable sex differences in the hippocampal phenotypes that emerge in adulthood after neonatal infection.

Early life infection alters adult BALB/c hippocampal gene expression in a sex specific manner.

During the perinatal period, the developing brain is sensitive to environmental events. Deleterious programing resulting from infection, dietary restriction, or psychological stress has been observed and affects adult immune and endocrine systems as well as behavior. In this study, we determined whether neonatal infection permanently alters immune and glucocorticoid receptor signaling pathways in the adult hippocampus. A Chlamydia muridarum respiratory infection was induced in male and female mice at birth. Mice were allowed to recover and microarray analysis was conducted on RNA from adult hippocampal tissue. In males, neonatal infection induced an up-regulation of genes associated with cellular development, nervous system development and function, such as cyclin-dependent kinase inhibitor 1A. After neonatal infection, adult females exhibited a T-helper 2 immune bias with genes such as major histocompatibility complex, class II, DQ beta 1 up-regulated. Expression of prolactin, vasopressin, hypocretin, corticotrophin-releasing hormone-binding protein, and oxytocin were confirmed by quantitative real-time polymerase chain reaction. This study shows that neonatal infection differentially alters the gene expression profiles of both female and male mice along immune and neuroendocrine pathways.

A pathogenic role for tumor necrosis factor-related apoptosis-inducing ligand in chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a life-threatening inflammatory respiratory disorder, often induced by cigarette smoke (CS) exposure. The development of effective therapies is impaired by a lack of understanding of the underlining mechanisms. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytokine with inflammatory and apoptotic properties. We interrogated a mouse model of CS-induced experimental COPD and human tissues to identify a novel role for TRAIL in COPD pathogenesis. CS exposure of wild-type mice increased TRAIL and its receptor messenger RNA (mRNA) expression and protein levels, as well as the number of TRAIL(+)CD11b(+) monocytes in the lung. TRAIL and its receptor mRNA were also increased in human COPD. CS-exposed TRAIL-deficient mice had decreased pulmonary inflammation, pro-inflammatory mediators, emphysema-like alveolar enlargement, and improved lung function. TRAIL-deficient mice also developed spontaneous small airway changes with increased epithelial cell thickness and collagen deposition, independent of CS exposure. Importantly, therapeutic neutralization of TRAIL, after the establishment of early-stage experimental COPD, reduced pulmonary inflammation, emphysema-like alveolar enlargement, and small airway changes. These data provide further evidence for TRAIL being a pivotal inflammatory factor in respiratory diseases, and the first preclinical evidence to suggest that therapeutic agents that target TRAIL may be effective in COPD therapy.

Mucosal production of uric acid by airway epithelial cells contributes to particulate matter-induced allergic sensitization.

Exposure to particulate matter (PM), a major component of air pollution, contributes to increased morbidity and mortality worldwide. PM induces innate immune responses and contributes to allergic sensitization, although the mechanisms governing this process remain unclear. Lung mucosal uric acid has also been linked to allergic sensitization. The links among PM exposure, uric acid, and allergic sensitization remain unexplored. We therefore investigated the mechanisms behind PM-induced allergic sensitization in the context of lung mucosal uric acid. PM10 and house dust mite exposure selectively induced lung mucosal uric acid production and secretion in vivo, which did not occur with other challenges (lipopolysaccharide, virus, bacteria, or inflammatory/fibrotic stimuli). PM10-induced uric acid mediates allergic sensitization and augments antigen-specific T-cell proliferation, which is inhibited by uricase. We then demonstrate that human airway epithelial cells secrete uric acid basally and after stimulation through a previously unidentified mucosal secretion system. Our work discovers a previously unknown mechanism of air pollution-induced, uric acid-mediated, allergic sensitization that may be important in the pathogenesis of asthma.

Co-transplantation of embryonic neural tissue and autologous peripheral nerve segments to severe spinal cord injury of the adult rat. Guided axogenesis from transplanted neurons.

The present study is the first of a series of experiments designed to investigate the possibilities of reconstructing the severely injured spinal cord by means of transplantation techniques. Special attention has been given here to the capability of transplanted embryonic neurons to extend axons into autologous peripheral nerve grafts (PNGs). A cavity, made unilaterally in the cervical enlargement of the spinal cord of adult rats, was filled with solid pieces of different embryonic tissues: spinal cord (SC), cortex (CT) or dorsal root ganglia (DRG). In more than half of the transplanted animals, one end of a PNG was inserted into the center of the transplants, while the other, extraspinal end, was crushed and tied to peripheral tissues. After a postgrafting period ranging from 1 to 6 months, we found that the 3 types of transplants in general had survived and become integrated with the host spinal cord, although their overall organization remained atypical. Surviving graft neurons had developed processes, some of which had become myelinated. The ability of the grafted neurons to extend axons into the PNG differed strikingly from one type of graft to another, being apparently non-existent for cortical grafts, moderate for spinal cord grafts and quite extensive for dorsal root ganglia transplants. Interestingly, these differences reflected what was observed for the corresponding, fully differentiated qeurons in adult animals, when their cut axons were put in contact with non-neuronal components of peripheral nerves.

Persistence of functional neuromuscular junctions formed in a denervated skeletal muscle of the adult rat by axons that have regrown from the injured spinal cord through a peripheral nerve autograft.

In previous 'short-term' (2 to 7 months) experiments, we had demonstrated, in the adult rat, that motoneurons of the injured cervical spinal cord could extend lengthy axons into an autologous peripheral nerve segment which was connected to a nearby denervated skeletal muscle. In addition, we had shown that new functional motor endplates were formed by these axons both at the original sites of innervation and at ectopic locations of the denervated muscle. This substitution motor system, although quite functional, was anatomically very different from the original model of innervation in the intact animal, relating to its motoneuronal pool, the course of its motor axons and the sites of terminal innervation. The present 'long-term' (11 to 21 months) experiments demonstrate the anatomical and functional permanency of the new motor circuitry, despite a lack of strict specificity in the new neuromuscular connections. However, some minor modifications or adjustments were observed with time: (i) the maintenance of functional ectopic endplates could be consistently demonstrated, while functional reinnervated endplates at the initial sites of innervation were rare or even lacking; (ii) there was a definitive withdrawal of all non target-specific regenerated axons from the vicinity of the muscle. It is now necessary to address the question to what extent this substitution motor system is actually controlled by central and/or peripheral inputs.

Expression of peripherin, NADPH-diaphorase and NOS in the adult rat neocortex.

Peripherin is mainly expressed in peripheral neurones and in CNS neurones which extend axons into peripheral nerves. However, this intermediate filament protein has also been detected in a few other neurones entirely located within the CNS. The present study focuses on the adult rat neocortex. Peripherin immunoreactive (P+) neuronal somata and their neuritic extensions were observed in cortical layers II, III, V and VI, while a few P+ nerve fibres could be seen in layer I. All the P+ neurones could be selectively stained using reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry, a typical feature of aspiny neurones. Some of the P+ neurones could also be immunostained with an antibody raised against nitric oxide synthase (NOS). These results provide evidence that peripherin is present in a discrete population of aspiny interneurones of the adult rat neocortex. The functional significance of the co-expression of peripherin and NOS needs further investigation.

Reinnervation of a denervated skeletal muscle by spinal axons regenerating through a collagen channel directly implanted into the rat spinal cord.

In the present study, the continuity between the central nervous system (CNS) and the peripheral nervous system (PNS) was restored by mean of a collagen channel in order to reinnervate a skeletal muscle. Three groups of animals were considered. In the first group, one end of the collagen channel was implanted in the cervical spinal cord of adult rats. The other end was connected to a 30-mm autologous peripheral nerve graft (PNG) implanted into the denervated biceps brachii muscle. The gap between the spinal cord and the proximal nerve stump varied from 3 to 7 mm. In the second group of animals, the distal end of the PNG graft was ligatured in order to compare the survival of the growing axons in the presence and in the absence of a muscular target. In the third group of animals, the extraspinal stump of the collagen channel was ligatured. Our study demonstrates that spinal neurons and dorsal root ganglion (DRG) neurons can grow long axons through the collagen channel over a 7-mm gap and reinnervate a denervated skeletal muscle. The results also indicate that the presence of a PNG at the extraspinal stump of the collagen channel is essential for axonal regrowth and that the muscle target contributes to the long-term maintenance of the regenerating axons. These data might be interesting for clinical application when the continuity between the CNS and PNS is interrupted such as in root avulsion.

Expression of peripherin in solid transplants of foetal spinal cord and dorsal root ganglia grafted to the injured cervical spinal cord of adult rats.

The expression of the neuronal type III intermediate filament protein peripherin was studied in E14 spinal cord fragments and E15 dorsal root ganglia 1-30 weeks after their transplantation to the injured cervical spinal cord of the adult rat. In the dorsal root ganglion transplants, the surviving neurons generally appeared as a rather healthy looking population of small strongly immunoreactive cells which are very similar to the small dorsal root ganglion neurons of adult control rats. In the spinal cord transplants, there were only a few peripherin-immunoreactive neurons, morphologically close to the motoneurons or to the preganglionic sympathetic neurons of adult rats. In both types of transplants, peripherin expression of the immunoreactive neurons was apparently correlated with the previously established ability of these transplanted neurons for extensive axonal growth into a co-grafted peripheral nerve.

Formation of functional endplates by spinal axons regenerating through a peripheral nerve graft. A study in the adult rat.

Peripheral nerve (PN) autografts were used in the adult rat to join the midcervical spinal cord to a nearby denervated skeletal muscle. Retrograde tracing, morphological and electrophysiological studies indicated the following: 1) a great number of neurons, located bilaterally, between C3 and C7 in most laminae of the grey matter, extended axons into the PN grafts, 2) a lesser number of neurons regenerated up to the reconnected muscle, but most of them were typical motoneurons, 3) neuromuscular junctions were formed in ectopic locations, around the tip of the grafted nerve, and at the sites of original endplates, 4) these junctions were functional and formed by axons that had regenerated into the PN bridges, as muscle contraction was obtained by electrical stimulation of the grafted nerves, 5) they were proved to be cholinergic since endplate potentials, evoked by stimulating the PN graft, were suppressed by curare. These results strongly suggest that spinal neurons, and especially motoneurons, are involved in the formation, through PN bridges, of new functional cholinergic connections with denervated skeletal muscles.

[Reconstruction of the spinal cord and its motor connections using embryonal nervous tissue transplantation and peripheral nerve autotransplantation. A study in the adult rat]. / Restructuration de la moelle épinière et de ses connexions motrices a l'aide de transplants de tissu nerveux embryonnaire et d'autogreffons de nerf périphérique. Etude chez le rat adulte.

Our research group is studying, in the adult rat, the conditions of an anatomical and functional reconstruction of the spinal cord and of its motor connections, following a spinal lesion that is either small (focal) or large (depletive). In this attempt to repair the damaged neuronal circuitry, we use, alone on in combination, two transplantation techniques, namely that of embryonic neural tissue, to replace the lost neurons, and that of long segments of autologous peripheral nerves to stimulate and guide either the axonal regrowth from injured host spinal neurons or the axogenesis of transplanted embryonic neurons. The common denominator to the whole experimentation is the setting up of a "nerve bridge" (peroneal nerve autograft) joining the injured cervical spinal cord an aneural region of a nearby denervated skeletal muscle. In a first experimental model (focal lesion), in which only a peripheral nerve autograft is used, it can be observed that local injured (or uninjured?) motoneurons have the actual capacity to extend axons throughout the nerve bridge and, thus, to reach the muscle and reform functional and stable, mainly ectopic, neuromuscular connections. In a second experimental model (depletion lesion) a cavity is made, by suction, in the cervical spinal cord, thus causing a damage which resembles, in some respects, certain types of neurodegenerative spinal lesions. This cavity is filled with different kinds of embryonic neural transplants. The surviving transplanted neurons differentiate axonal projections, some of them extending into the peripheral nerve bridge. Studies aimed at determining the capacities of motor endplate formation by the axons that have grown from these neurons of substitution throughout the nerve bridge, as well as the possibilities of reafferentiation of the transplanted tissues by regenerating host "central" nerve fibres, are in progress.

[Spinal cord reconstruction and neural transplants. New therapeutic vectors]. / Restructuration médullaire et transplants nerveux. Nouveaux vecteurs thérapeutiques.

Spontaneous recovery from severe traumatic lesions of the central nervous system (CNS) does not occur in adult mammals for two main reasons: the lost neurons are not replaced and the surviving axotomized nerve cells do not regenerate the missing part of their cut axon, thus failing to reestablish the original anatomical and functional connections. The likeliest explanation for this lack of axonal regrowth is powerful inhibitory effects from mature reactive astrocytes and oligodendrocytes. However, experimental strategies making use of transplantation techniques have proved to be rather efficient. Thus, with regard to spinal cord injury: 1) lost spinal neurons (especially motoneurons) can, to some extent, be replaced by foetal spinal neurons transplanted to the lesion site; 2) irreversible damage to spinal projections from supraspinal neurons (mainly localized in the brain stem) can partially be compensated by grafting homologous foetal nerve cells caudally with regard to the spinal lesion site; 3) axogenesis from transplanted nerve cells or axonal regrowth from injured host neurons can be triggered and guided towards central or peripheral targets by autologous peripheral nerve segments which bypass the lesion site. This efficient cellular therapy is on the way to be complemented by a gene therapy that will allow the introduction, into the brain and the spinal cord, either of genetically modified cells from various origins, or of selected beneficial genes previously integrated into viral vectors.

Tumor necrosis factor-related apoptosis-inducing ligand translates neonatal respiratory infection into chronic lung disease.

Respiratory infections in early life can lead to chronic respiratory disease. Chlamydia infections are common causes of respiratory disease, particularly pneumonia in neonates, and are linked to permanent reductions in pulmonary function and the induction of asthma. However, the immune responses that protect against early-life infection and the mechanisms that lead to chronic lung disease are incompletely understood. Here we identify novel roles for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in promoting Chlamydia respiratory infection-induced pathology in early life, and subsequent chronic lung disease. By infecting TRAIL-deficient neonatal mice and using neutralizing antibodies against this factor and its receptors in wild-type mice, we demonstrate that TRAIL is critical in promoting infection-induced histopathology, inflammation, and mucus hypersecretion, as well as subsequent alveolar enlargement and impaired lung function. This suggests that therapeutic agents that target TRAIL or its receptors may be effective treatments for early-life respiratory infections and associated chronic lung disease.

Constitutive production of IL-13 promotes early-life Chlamydia respiratory infection and allergic airway disease.

Deleterious responses to pathogens during infancy may contribute to infection and associated asthma. Chlamydia respiratory infections in early life are common causes of pneumonia and lead to reduced lung function and asthma. We investigated the role of interleukin-13 (IL-13) in promoting early-life Chlamydia respiratory infection, infection-induced airway hyperresponsiveness (AHR), and severe allergic airway disease (AAD). Infected infant Il13(-/-) mice had reduced infection, inflammation, and mucus-secreting cell hyperplasia. Surprisingly, infection of wild-type (WT) mice did not increase IL-13 production but reduced IL-13Rα2 decoy receptor levels compared with sham-inoculated controls. Infection of WT but not Il13(-/-) mice induced persistent AHR. Infection and associated pathology were restored in infected Il13(-/-) mice by reconstitution with IL-13. Stat6(-/-) mice were also largely protected. Neutralization of IL-13 during infection prevented subsequent infection-induced severe AAD. Thus, early-life Chlamydia respiratory infection reduces IL-13Rα2 production, which may enhance the effects of constitutive IL-13 and promote more severe infection, persistent AHR, and AAD.

Programming of the lung by early-life infection.

Many important human diseases, such as asthma, have their developmental origins in early life. Respiratory infections in particular may alter the course of asthma and may either protect against or promote the development of this disease. It is likely that the nature of the effects depends on the type and age of infection and is determined by the impact of infection on the immune and respiratory systems. Immunity in early life is plastic and can be moulded by antigen encounter, which may enhance or reinforce the asthmatic phenotype of early life, or induce protective responses. Chlamydial respiratory infections have specific effects and may increase asthma severity in early life by promoting systemic interleukin 13 responses and causing permanent changes in lung structure. Respiratory viral infections, such as those of respiratory syncytial virus and rhinovirus, promote pro-asthmatic responses in early life that contribute to the induction of asthma. By contrast, probiotics or infection or exposure to certain bacteria, such as Streptococcus pneumoniae, may have protective effects in asthma by increasing the numbers and activity of regulatory T cells. Here, we review the impact of infections on the developmental origins of asthma. Understanding these effects may lead to new therapeutic approaches for asthma that either target deleterious infections or utilize beneficial ones.