Characterization of Epstein-Barr virus-specific immune responses and of the novel cytokine interleukin-26 in patients with multiple sclerosis

RÉSUMÉ La sclérose en plaques (SEP) est une maladie démyélinisante du système nerveux central (SNC) qui touche le plus souvent de jeunes femmes. Bien qu'elle ait été décrite pour la première fois il y a plus de 200 ans, son étiologie n'est pas encore complètement comprise. Contrairement à d'autres maladies purement génétiques, l'épidémiologie de la SEP ne peut être que partiellement expliquée par des facteurs génétiques. Ceci suggère que des facteurs environnementaux pourraient être impliqués dans la pathogenèse de la SEP. Parmi ceux-ci, le virus d'Epstein-Barr (EBV) est un excellent candidat, comme cela a été démontré par de larges études séroépidémiologiques ainsi que pax l'évaluation de la réponse cellulaire dans le sang. Bien que le SNC soit en fait la cible des réponses immunitaires anormales dans la SEP, peu d'études ont été accomplies sur les réponses immunitaires spécifiques à EBV dans ce compartiment. Ceci est particulièrement vrai chez des patients vivants chez lesquels des biopsies sont rarement effectuées, ainsi que pour les réponses cellulaires car très peu de cellules immunitaires peuvent être obtenues du SNC. Nous avons donc développé des conditions de cultures et un readout nous permettant d'étudier le nombre réduit de cellules disponibles dans le liquide céphalo-rachidien (LCR), qui représente le seul matériel pouvant être obtenu du SNC de patients SEP vivants. Nous avons trouvé que les réponses cellulaires et humorales spécifiques à EBV étaient augmentées dans le LCR des patients SEP comparé à du sang pairé, ainsi que par rapport à des patients avec d'autres maladies neurologiques inflammatoires et noninflammatoires. Afin de déterminer si les réponses immunitaires augmentées contre EBV étaient spécifiques à ce virus ou si elles reflétaient simplement une hyperactivation immunitaire aspécifique, nous avons comparé les réponses spécifiques à EBV avec celles spécifiques au cytomegalovirus (CNN). En effet, comme EBV, CNN est un herpesvirus neurotropique qui peut établir des infections latentes, mais ce dernier n'est pas considéré comme étant associé à la SEP. De façon intéressante, les réponses immunitaires spécifiques à CNN trouvées dans le LCR étaient plus basses que dans le sang, et ceci dans toutes les catégories de patients. Ces données suggèrent qu'une réactivation d'EBV pourrait avoir lieu dans le SNC des patients SEP à un stade précoce de la maladie et renforcent fortement l'hypothèse qu'EBV pourrait avoir un rôle déclencheur dans cette maladie. Ainsi, il pourrait être intéressant d'explorer si un traitement ou un vaccin efficace contre EBV peut prévenir le développement de la SEP. On ne connaît toujours pas la raison pour laquelle les réponses immunitaires spécifiques à EBV sont augmentées chez les patients SEP. Une hypothèse est que la réponse immunitaire est qualitativement différente chez les patients SEP par rapports aux contrôles. Pour examiner ceci, nous avons évalué le profile cytokinique de lymphocytes T CD4+ et CD8+ stimulés par EBV, mais nous n'avons pas pu mettre en évidence de différence remarquable entre patients SEP et sujets sains. Cette question reste donc ouverte et d'autres études sont justifiées. Il n'existe pas de marqueur fiable de la SEP. Ici, nous avons trouvé que la cytokine IL-26, récemment décrite, était augmentée dans les lymphocytes T CD8+ des patients avec une SEP secondairement progressive comparé à des patients SEP en poussée, des patients avec une SEP primairement progressive, des patients avec d'autres maladies neurologiques inflammatoires, ou des sujets sains. De plus, nous avons identifié des types de cellules dérivées du cerveau (astrocytes, oligodendrocytes et neurones) qui exprimaient le récepteur de l'IL-26. Ceci ouvre la voie à d'autres études afin de mieux comprendre la fonction de l'II.-26 et son interaction avec la. SEP. SUMMARY : Multiple sclerosis (MS) is a demyelinating disease affecting the central nervous system (CNS), mostly in young female adults. Although it was first described 200 years ago, its etiology is still not completely understood. Contrary to other purely genetic diseases, genetics can explain only part of MS epidemiology. Therefore, environmental factors that might be involved in MS pathogenesis were searched for. Among them, Epstein-Barr virus (EBV) is a strong potential candidate, such as shown by large seroepidemiological studies and cellular immune response assessments in the blood. Although the CNS is the actual target of abnormal immune responses in MS, few studies have been performed on EBV-specific immune responses in this compartment. This is particularly true for live patients, from which biopsy material is almost never available, and for cellular immune responses, since very few immune cells are available from the CNS. We therefore developed culture conditions and a readout that were compatible with the study of the reduced number of cells found in the cerebrospinal fluid (CSF), the only readily available material from the CNS of live ' MS patients. We found that EBV-specific cellular and humoral immune responses were increased in the CSF of MS patients as compared with paired blood, as well as compared with the CSF of patients with other inflammatory and non-inflammatory neurological diseases. To determine whether the enhanced immune responses against EBV were specific of this virus or simply reflected an aspecific immune hyperactivation, we compared the EBV- with the cytomegalovirus (CMV)-specific immune responses. Indeed, like EBV, CMV is a neurotrophic herpesvirus that can establish latent infections, but the latter is not considered to be associated with MS. Interestingly, CSF CMV-specific immune responses were lower than blood ones and this, in all patient categories. These findings suggest that EBV reactivation may be taking place in the CNS of patients at the early stages of MS and strengthen the hypothesis that EBV may have a triggering role in this disease. Therefore, it might be interesting to explore whether an efficient anti-EBV drug or vaccine is able to prevent MS development. The reason why EBV-specific immune responses are increased in MS patients is still missing. One hypothesis might be that the immune response against EBV is qualitatively different in MS patients as compared with controls. To examine this, we assessed the cytokine mRNA profile of EBV-stimulated CD4+ and CD8+ T cells, but could not find any remarkable difference between MS patients and healthy controls. Therefore, this question remains open and fiirther studies are warranted. Reliable disease markers are lacking for MS. Here, we found that the recently described cytokine IL-26 was increased in CD8+ T cells of patients with secondary progressive MS as compared with relapsing MS, primary progressive MS, other inflammatory neurological diseases and healthy controls. Moreover, we identified brain cell types (astrocytes, oligodendrocytes and neurons) that expressed the IL-26 receptor, paring the way for further studies to understand IL-26 function and its interaction with MS.

Condition, disease and immune defence

Life-history traits and secondary sexual characters often demonstrate condition-dependence, and reproductive success thus ultimately appears to be determined by condition. Here we test the hypothesis that anti-parasite defence is condition-dependent and thus ultimately limits fitness. Animal hosts defend themselves against parasites by an efficient immune system that changes its activity level depending on level of infection. Since immune function is costly, as demonstrated by several field studies, we predicted that large immune defence organs should be maintained when the costs of an elevated immune response were reduced, or when the benefits were increased. Hence, the size of immune defence organs was predicted to increase in response to disease due to increased benefits of investment in immune function, and the; size was predicted to increase in response to high body condition because of reduced costs of investment in immune function. A comparative study of birds demonstrated that the size of the spleen was significantly increased among individuals suffering from parasitic infections and signs of disease as compared to healthy individuals. Furthermore, we found evidence for a positive association between spleen size and body condition. These findings are consistent with the hypothesised cost of immune function and hence a cost of anti-parasite defence.

IL-28A (IFN-λ2) modulates lung DC function to promote Th1 immune skewing and suppress allergic airway disease.

IL-28 (IFN-λ) cytokines exhibit potent antiviral and antitumor function but their full spectrum of activities remains largely unknown. Recently, IL-28 cytokine family members were found to be profoundly down-regulated in allergic asthma. We now reveal a novel role of IL-28 cytokines in inducing type 1 immunity and protection from allergic airway disease. Treatment of wild-type mice with recombinant or adenovirally expressed IL-28A ameliorated allergic airway disease, suppressed Th2 and Th17 responses and induced IFN-γ. Moreover, abrogation of endogenous IL-28 cytokine function in IL-28Rα(-/-) mice exacerbated allergic airway inflammation by augmenting Th2 and Th17 responses, and IgE levels. Central to IL-28A immunoregulatory activity was its capacity to modulate lung CD11c(+) dendritic cell (DC) function to down-regulate OX40L, up-regulate IL-12p70 and promote Th1 differentiation. Consistently, IL-28A-mediated protection was absent in IFN-γ(-/-) mice or after IL-12 neutralization and could be adoptively transferred by IL-28A-treated CD11c(+) cells. These data demonstrate a critical role of IL-28 cytokines in controlling T cell responses in vivo through the modulation of lung CD11c(+) DC function in experimental allergic asthma. →See accompanying Closeup by Michael R Edwards and Sebastian L Johnston http://dx.doi.org/10.1002/emmm.201100143.

Inflammasome activation by adenylate cyclase toxin directs Th17 responses and protection against Bordetella pertussis.

Inflammasome-mediated IL-1beta production is central to the innate immune defects that give rise to certain autoinflammatory diseases and may also be associated with the generation of IL-17-producing CD4(+) T (Th17) cells that mediate autoimmunity. However, the role of the inflammasome in driving adaptive immunity to infection has not been addressed. In this article, we demonstrate that inflammasome-mediated IL-1beta plays a critical role in promoting Ag-specific Th17 cells and in generating protective immunity against Bordetella pertussis infection. Using a murine respiratory challenge model, we demonstrated that the course of B. pertussis infection was significantly exacerbated in IL-1R type I-defective (IL-1RI(-/-)) mice. We found that adenylate cyclase toxin (CyaA), a key virulence factor secreted by B. pertussis, induced robust IL-1beta production by dendritic cells through activation of caspase-1 and the NALP3-containing inflammasome complex. Using mutant toxins, we demonstrate that CyaA-mediated activation of caspase-1 was not dependent on adenylate cyclase enzyme activity but was dependent on the pore-forming capacity of CyaA. In addition, CyaA promoted the induction of Ag-specific Th17 cells in wild-type but not IL-1RI(-/-) mice. Furthermore, the bacterial load was enhanced in IL-17-defective mice. Our findings demonstrate that CyaA, a virulence factor from B. pertussis, promotes innate IL-1beta production via activation of the NALP3 inflammasome and, thereby, polarizes T cell responses toward the Th17 subtype. In addition to its known role in subverting host immunity, our findings suggest that CyaA can promote IL-1beta-mediated Th17 cells, which promote clearance of the bacteria from the respiratory tract.

Meningococcal disease in a kidney transplant recipient with mannose-binding lectin (MBL) deficiency

Background: There is an increasing amount of data associating MBL deficiency with a higher susceptibility to meningococca[ disease. In addition, meningococca[ disease has been reported in patients with various immunosuppressive conditions. However, to our knowledge, only three cases of meningococca[ disease have been reported in solid organ recipients (SOT). Methods & Results: A 32 year-old male patient underwent cadaveric kidney transplantation for endstage renal disease of unknown origin. On day 71 post-transplantation he developed fever (39.6°C), shaking chilis, and tachycardia without hypotension. At this time, immunosuppression consisted of tacro[imus, prednisone 10mg daily and mycopheno[ ate mofeti[ 2 g daily. Physical examination on admission was normal, except for two small petechia[ lesions on the forearm. No meningeal signs were present. Three sets of blood cultures grew Neisseria meningitidis group C susceptible to ceftriaxone (MIC=0.003mg/[). Antibiotic therapy consisted in intravenous ceftriaxone 2 g per day for a total duration of 7 days. Serum immunog[obu[in levels, C3, C4 and CHS0 were normal However, using a method to screen for the functional activity of a[[ three pathways of complement (Wies[ab, Lund, Sweden), no activation via the MBL pathway could be detected (0%). A subsequent quantification of MBL pathway components revealed normal levels of MASP 2 but undetectab[e amounts of MBL (below 10 ng/m[, normal range: >500 ng/m[). Conclusion: Since the exact incidence and the possible relationship between meningococca[ disease and organ transplantation is not we[[ understood, we strongly encourage transplantation centers to report additional cases. The potential clinical usefu[ ness of screening SOT candidates for MBL deficiency in relation to infectious complications after transplantation remains to be determined.

Glut-1 deficiency syndrome masquerading as idiopathic generalized epilepsy.

To report the case of a child with short absences and occasional myoclonias since infancy who was first diagnosed with an idiopathic generalized epilepsy, but was documented at follow-up to have a mild phenotype of glucose transporter type 1 deficiency syndrome. Unlike other reported cases of Glut-1 DS and epilepsy, this child had a normal development as well as a normal head growth and neurological examination. Early onset of seizures and later recognized episodes of mild confusion before meals together with persistent atypical EEG features and unexpected learning difficulties led to the diagnosis. Seizure control and neuropsychological improvements were obtained with a ketogenic diet.

Regulatory B cells shape the development of Th2 immune responses in BALB/c mice infected with Leishmania major through IL-10 production.

Recent evidence indicates that B cells are required for susceptibility to infection with Leishmania major in BALB/c mice. In this study, we analyzed the role of the IL-10 produced by B cells in this process. We showed that B cells purified from the spleen of BALB/c mice produced IL-10 in response to stimulation with L. major in vitro. In vivo, early IL-10 mRNA expression is detected after L. major infection in B cells from draining lymph nodes of susceptible BALB/c, but not of resistant C57BL/6 mice. Although adoptive transfer of naive wild-type B cells prior to infection in B cell-deficient BALB/c mice restored Th2 cell development and susceptibility to infection with L. major of these otherwise resistant mice, adoptive transfer of IL-10(-/-) B cells mice did not. B cells stimulated by L. major, following in vitro or in vivo encounter, express the CD1d and CD5 molecules and the IL-10 produced by these cells downregulate IL-12 production by L. major-stimulated dendritic cells. These observations indicate that IL-10 secreting B cells are phenotypically and functionally regulatory B cells. Altogether these results demonstrate that the IL-10 produced by regulatory CD1d+ CD5+ B cells in response to L. major is critical for Th2 cell development in BALB/c mice.

Secreted glutamic protease rescues aspartic protease Pep deficiency in Aspergillus fumigatus during growth in acidic protein medium.

In an acidic protein medium Aspergillus fumigatus secretes an aspartic endoprotease (Pep) as well as tripeptidyl-peptidases, a prolyl-peptidase and carboxypeptidases. In addition, LC-MS/MS revealed a novel glutamic protease, AfuGprA, homologous to Aspergillus niger aspergillopepsin II. The importance of AfuGprA in protein digestion was evaluated by deletion of its encoding gene in A. fumigatus wild-type D141 and in a pepΔ mutant. Either A. fumigatus Pep or AfuGprA was shown to be necessary for fungal growth in protein medium at low pH. Exoproteolytic activity is therefore not sufficient for complete protein hydrolysis and fungal growth in a medium containing proteins as the sole nitrogen source. Pep and AfuGprA constitute a pair of endoproteases active at low pH, in analogy to A. fumigatus alkaline protease (Alp) and metalloprotease I (Mep), where at least one of these enzymes is necessary for fungal growth in protein medium at neutral pH. Heterologous expression of AfuGprA in Pichia pastoris showed that the enzyme is synthesized as a preproprotein and that the propeptide is removed through an autoproteolytic reaction at low pH to generate the mature protease. In contrast to A. niger aspergillopepsin II, AfuGprA is a single-chain protein and is structurally more similar to G1 proteases characterized in other non-Aspergillus fungi.

Acute coronary syndrome after levamisole-adultered cocaine abuse

Cocaine is a well known trigger of acute coronary syndromes. Over the last 10 years levamisole, a veterinary anthelminthic drug has been increasingly used as an adulterant of cocaine. Levamisole was used to treat pediatric nephritic syndrome and rheumatoid arthritis before being withdrawn from the market due to its significant toxicity, i.e. hematological complications and vasculitis. The major complications of levamisole-adultered cocaine reported up to now are hematological and dermatological. The case reported here is of a 25 year old man with a history of cocaine abuse who died at home after complaining of retrosternal pain. Postmortem CT-angiography, autopsy, and chemical and toxicological analyses were performed. An eroded coronary artery plaque was found at the proximal segment of the left anterior descending coronary artery. Two myocardial infarct scars were present in the left ventricle. Microscopic examination of the coronary artery revealed infiltration of eosinophils into the adventitia and intima. Toxicological examination confirmed the presence of cocaine and its metabolites in the peripheral blood, and of levamisole in the urine and pericardial fluid. Eosinophilic inflammatory coronary artery pathologies have been clinically linked to coronary dissection, hypersensitivity coronary syndrome and vasospastic allergic angina. The coronary pathology in the presented case could be a complication of levamisole-adultered cocaine use, in which an allergic or immune-mediated mechanism might play a role. The rise in cocaine addiction worldwide and the increase of levamisole adulterated cocaine highlights the importance of updating our knowledge of the effects of adultered cocaine abuse.

Pre-hatching maternal effects inhibit nestling humoral immune response in the tawny owl Strix aluco

Although evidence is accumulating that mothers can transfer antibodies to their offspring, little is known about the consequences of such a transfer to the offspring immune system. Because maternal antibodies are effective only during a short period of time after their transfer to offspring, one hypothesis is that maternal antibodies provides a transitory antigen-specific protection to offspring, thus lessening the need for offspring to mount their own humoral immune response towards these specific antigens. In birds, this scenario predicts that offspring immune response towards a specific antigen is inhibited to a larger extent in hatchlings than in older nestlings. We tested this hypothesis in tawny owls Strix aluco by cross-fostering clutches between nests and then challenging siblings with a vaccine either two times (at 4- and 11-d-old) or only one time at 11-d-old to compare the strength of the humoral response between nestlings born from mothers with naturally high and low levels of antibodies against this vaccine. Because maternal antibodies are expected to be effective only during a short period of time after hatching, we predict that maternal antibodies should inhibit the immune response of nestlings vaccinated from the fourth day after hatching more than in nestlings vaccinated only at a later age. As expected, the inhibitory effect of maternal antibodies was stronger in nestlings vaccinated soon after hatching than in siblings injected at a later age. Therefore, in wild avian populations pre-hatching maternal effects may confer offspring with a transitory immune protection in the first days following hatching.

Activated group 3 innate lymphoid cells promote T-cell-mediated immune responses.

Group 3 innate lymphoid cells (ILC3s) have emerged as important cellular players in tissue repair and innate immunity. Whether these cells meaningfully regulate adaptive immune responses upon activation has yet to be explored. Here we show that upon IL-1β stimulation, peripheral ILC3s become activated, secrete cytokines, up-regulate surface MHC class II molecules, and express costimulatory molecules. ILC3s can take up latex beads, process protein antigen, and consequently prime CD4(+) T-cell responses in vitro. The cognate interaction of ILC3s and CD4(+) T cells leads to T-cell proliferation both in vitro and in vivo, whereas its disruption impairs specific T-cell and T-dependent B-cell responses in vivo. In addition, the ILC3-CD4(+) T-cell interaction is bidirectional and leads to the activation of ILC3s. Taken together, our data reveal a novel activation-dependent function of peripheral ILC3s in eliciting cognate CD4(+) T-cell immune responses.

The influence of environmental conditions on immune responses, morphology and recapture probability of nestling house martins (Delichon urbica)

Nestling birds produced later in the season are hypothesized to be of poor quality with a low probability of survival and recruitment. In a Spanish population of house martins (Delichon urbica), we first compared reproductive success, immune responses and morphological traits between the first and the second broods. Second, we investigated the effects of an ectoparasite treatment and breeding date on the recapture rate the following year. Due probably to a reverse situation in weather conditions during the experiment, with more rain during rearing of the first brood, nestlings reared during the second brood were in better condition and had stronger immune responses compared with nestlings from the first brood. Contrary to other findings on house martins, we found a similar recapture rate for chicks reared during the first and the second brood. Furthermore, ectoparasitic house martin bugs had no significant effect on the recapture rate. Recaptured birds had similar morphology but higher immunoglobulin levels when nestlings compared with non-recaptured birds. This result implies that a measure of immune function is a better predictor of survival than body condition per se.