FOXP3 Expression Is Upregulated in CD4+T Cells in Progressive HIV-1 Infection and Is a Marker of Disease Severity

Understanding the role of different classes of T cells during HIV infection is critical to determining which responses correlate with protective immunity. To date, it is unclear whether alterations in regulatory T cell (Treg) function are contributory to progression of HIV infection.FOXP3 expression was measured by both qRT-PCR and by flow cytometry in HIV-infected individuals and uninfected controls together with expression of CD25, GITR and CTLA-4. Cultured peripheral blood mononuclear cells were stimulated with anti-CD3 and cell proliferation was assessed by CFSE dilution.HIV infected individuals had significantly higher frequencies of CD4(+)FOXP3(+) T cells (median of 8.11%; range 1.33%-26.27%) than healthy controls (median 3.72%; range 1.3-7.5%; P = 0.002), despite having lower absolute counts of CD4(+)FOXP3(+) T cells. There was a significant positive correlation between the frequency of CD4(+)FOXP3(+) T cells and viral load (rho = 0.593 P = 0.003) and a significant negative correlation with CD4 count (rho = -0.423 P = 0.044). 48% of our patients had CD4 counts below 200 cells/microl and these patients showed a marked elevation of FOXP3 percentage (median 10% range 4.07%-26.27%). Assessing the mechanism of increased FOXP3 frequency, we found that the high FOXP3 levels noted in HIV infected individuals dropped rapidly in unstimulated culture conditions but could be restimulated by T cell receptor stimulation. This suggests that the high FOXP3 expression in HIV infected patients is likely due to FOXP3 upregulation by individual CD4(+) T cells following antigenic or other stimulation.FOXP3 expression in the CD4(+) T cell population is a marker of severity of HIV infection and a potential prognostic marker of disease progression

Le gain de force du muscle lisse des voies aériennes dans l'asthme : une étude translationnelle

L’asthme est un désordre respiratoire obstructif qui affecte plus de 330 millions de personnes à travers le monde. Les symptômes de cette pathologie comprennent de l’essoufflement, de l’oppression thoracique, de la sibilance et de la toux, et surviennent suivant l’inhalation de facteurs déclencheurs (virus, allergène, pollution…). La pathologie de l’asthme est caractérisée par une inflammation chronique et variable au sein du système respiratoire, un remodelage des voies aériennes ainsi qu’une hyperréactivité bronchique. L’équipe de recherche du Dr Ynuk Bossé travaille sur la physiologie du muscle lisse des voies aériennes, et plus spécifiquement sur l’augmentation des capacités contractiles du muscle lisse en réponse à un tonus (i.e. contraction soutenue). Ce phénomène nommé le gain de force du muscle lisse a été observé avant mon arrivée dans ce laboratoire sur des trachées de moutons et de souris montées en bain d’organe, ainsi qu’in vivo chez la souris. Des travaux antérieurs ont démontré que la présence d’un tonus augmentait la réactivité bronchique de souris en réponse à l’inhalation d’une forte dose de métacholine. Le but de cette thèse était d’explorer dans une dynamique translationnelle, les rouages moléculaires de ce phénomène et d’en définir les impacts sur la fonction respiratoire in vivo. Dans une première étude réalisée au début de mon doctorat, nous avons étudié les conséquences du tonus sur la réactivité bronchique in vivo chez l’humain. Nous avons ainsi observé que l’augmentation du tonus, provoquée par l’inhalation répétée de faibles doses de métacholine durant une période de 30 minutes, augmentait la réactivité bronchique en réponse à l’inhalation d’une forte dose de métacholine. De plus, nous avons également observé grâce à l’utilisation de la technique des oscillations forcées que cette augmentation de la réactivité bronchique était liée à une augmentation de la résistance des voies périphériques. Nous avons donc confirmé dans cette étude que la présence d’un tonus augmente la réactivité bronchique chez de jeunes humains en santé. Dans une seconde étude, conduite tout au long de mon doctorat, nous avons étudié les mécanismes moléculaires responsables du gain de force du muscle lisse en réponse à un tonus. Nous avons ainsi observé que ce phénomène n’était pas lié à une potentialisation de la phosphorylation de la chaîne légère de myosine, mais plutôt provoqué par une augmentation de la filamentogénèse d’actine. Nous avons également déterminé que cette augmentation de la filamentogénèse d’actine était en partie provoquée par une inhibition de la dépolymérisation des filaments d’actine suite à l’inhibition de la protéine cofiline. Nous avons donc démontré que le tonus augmente la filamentogénèse d’actine au sein des cellules musculaires lisses, ce qui pourrait contribuer à une augmentation des capacités contractiles. Finalement, dans une troisième étude entreprise durant la dernière partie de mon doctorat, nous tentons de comprendre les liens entre le phénomène du gain de force du muscle lisse et l’inflammation présente dans l’asthme. Il semblerait que la présence d’une inflammation provoquée par de la poudre d’acariens chez la souris augmente la réactivité bronchique, mais empêche le développement du gain de force du muscle lisse. En revanche, les résultats obtenus sont encore préliminaires. Il est actuellement impossible de tirer des conclusions fermes. Ainsi, nous nous interrogeons toujours à l’égard du rôle de l’inflammation sur le gain de force du muscle lisse des voies aériennes. Dans sa globalité, cette thèse démontre que le gain de force du muscle lisse des voies aériennes est provoqué par un remodelage du cytosquelette d’actine, et que ce phénomène augmente la réactivité bronchique in vivo chez l’humain. De plus, cette thèse ouvre des voies de recherche afin de déterminer si ce phénomène pourrait être impliqué dans l’hyperréactivité bronchique dans l’asthme.Asthma is an obstructive respiratory disorder affecting more than 330 million people worldwide. The symptoms include breathlessness, chest oppression, wheezing and cough. The symptoms are variable in nature and severity and generally coincide with the inhalation of environmental factors (viruses, allergens, pollution…). The pathology of asthma is characterized by several typical features, such as airway inflammation, airway remodeling and airway hyperresponsiveness. The research team of Dr Ynuk Bossé is specialized in the study of lung physiology and airway smooth muscle mechanics. Of particular interest is a phenomenon called ‘force adaptation’. Force adaptation is a time-dependent gain in the contractile capacity of airway smooth muscle in response to tone (i.e., a sustained contraction). This phenomenon was observed in vitro in isolated ovine and murine tissues, as well as in vivo in mice. Previous work has demonstrated that the presence of tone, provoked by repeated exposures to low doses of methacholine during 20 min, increases airway responsiveness to the inhalation of a high dose of methacholine. The aim of this thesis was to decipher the molecular mechanisms of force adaptation in vitro and to explore the impact of this phenomenon on respiratory function in vivo. In a first study, which was conducted at the beginning of my PhD, we assessed the effect of tone on airway responsiveness in young healthy adults. We demonstrated that tone, which was generated by repeated inhalations of low doses of methacholine during 30 min, enhances airway responsiveness to a high dose of methacholine. Moreover, with the use of the force oscillation technique, we demonstrated that this effect was predominant in the peripheral airways. Therefore, this study confirmed that airway smooth muscle tone increases airway responsiveness in young healthy adults. In a second study, conducted over the entire course of my PhD, we investigated the molecular mechanisms responsible for the gain in contractile capacity induced by tone. We observed that force adaptation does not rely on molecular mechanisms enhancing the phosphorylation of the myosin light chain but rather occurs in conjunction with an increase in actin filamentogenesis. We further demonstrated that this increase in actin filamentogenesis may stem not only from actin polymerization but also from the inhibition of actin filament depolymerization via the inhibition of the protein cofilin. Therefore, the results of this study suggested that tone increase the contractile capacity of airway smooth muscle by fostering actin filamentogenesis. Finally, in a third study started at the end of my PhD, we are trying to understand the links between the gain in contractile capacity induced by tone and airway inflammation in asthma. We are using a mouse model of allergic airway inflammation induced by repeated exposures to house dust mite. While allergic inflammation increases airway responsiveness, it seems to diminish the phenomenon of force adaptation. However, the results obtained so far will require further investigations. It is currently impossible to reach authoritative conclusions. We are still left wondering whether airway inflammation alters the gain in contractile capacity induced by tone. Overall, this thesis is demonstrated that force adaptation increases airway responsiveness in vivo in human and, at the molecular level, the phenomenon seems to rely on an active remodeling of the actin cytoskeleton. Moreover, this thesis opens new research areas, which will need to be further explored in order to determine whether the gain in contractile capacity induced by tone is implicated in airway hyperresponsiveness in asthm

Unlocking the Complexity of Neuromuscular Diseases: Insights from Human Pluripotent Stem Cell-Derived Neuromuscular Junctions

Over the past 20 years, the use of pluripotent stem cells to mimic the complexities of the human neuromuscular junction has received much attention. Deciphering the key mechanisms underlying the establishment and maturation of this complex synapse has been driven by the dual goals of addressing developmental questions and gaining insight into neuromuscular disorders. This review aims to summarise the evolution and sophistication of in vitro neuromuscular junction models developed from the first differentiation of human embryonic stem cells into motor neurons to recent neuromuscular organoids. We also discuss the potential offered by these models to decipher different neuromuscular diseases characterised by defects in the presynaptic compartment, the neuromuscular junction, and the postsynaptic compartment. Finally, we discuss the emerging field that considers the use of these techniques in drug screening assay and the challenges they will face in the future

The underlying physiological mechanisms whereby anti-cholinergics alleviate asthma

The mechanisms whereby anti-cholinergics improve asthma outcomes, such as lung function, symptoms and rate of exacerbation, can be numerous. The most obvious is by affecting the contraction of airway smooth muscle (ASM). The acetylcholine released from the cholinergic nerves is the most important bronchoconstrictor that sets the baseline degree of contractile activation of ASM in healthy individuals. Although the degree of ASMâ s contractile activation can also be fine-tuned by a plethora of other bronchoconstrictors and bronchodilators in asthma, blocking the ceaseless effect of acetylcholine on ASM by anti-cholinergics reduces, at any given moment, the overall degree of contractile activation. Since the relationships that exist between the degree of contractile activation, ASM force, ASM shortening, airway narrowing, airflow resistance and respiratory resistance are not linear, small decreases in the contractile activation of ASM can be greatly amplified and thus translate into important benefits on patientâ s well-being. Plus, many inflammatory and remodeling features that are often found in asthmatic lungs synergize with the contractile activation of ASM to increase respiratory resistance. This review recalls that the proven effectiveness of anti-cholinergics in the treatment of asthma could be merely attributed to a small reduction in the contractile activation of ASM.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Airway smooth muscle adapting in dynamic conditions is refractory to the bronchodilator effect of a deep inspiration

Airway smooth muscle (ASM) is continuously strained during breathing at tidal volume. Whether this tidal strain influences the magnitude of the bronchodilator response to a deep inspiration (DI) is not clearly defined. The present in vitro study examines the effect of tidal strain on the bronchodilator effect of DIs. ASM strips from sheep tracheas were mounted in organ baths and then subjected to stretches (30% strain), simulating DIs at varying time intervals. In between simulated DIs, the strips were either held at a fixed length (isometric) or oscillated continuously by 6% (length oscillations) to simulate tidal strain. The contractile state of the strips was also controlled by adding either methacholine or isoproterenol to activate or relax ASM, respectively. Although the time-dependent gain in force caused by methacholine was attenuated by length oscillations, part of the acquired force in the oscillating condition was preserved postsimulated DIs, which was not the case in the isometric condition. Consequently, the bronchodilator effect of simulated DIs (i.e., the decline in force postsimulated versus presimulated DIs) was attenuated in oscillating versus isometric conditions. These findings suggest that an ASM operating in a dynamic environment acquired adaptations that make it refractory to the decline in contractility inflicted by a larger strain simulating a DI

Shortening of airway smooth muscle is modulated by prolonging the time without simulated deep inspirations in ovine tracheal strips

The shortening of airway smooth muscle (ASM) is greatly affected by time. This is because stimuli affecting ASM shortening, such as bronchoactive molecules or the strain inflicted by breathing maneuvers, not only alter quick biochemical processes regulating contraction but also slower processes that allow ASM to adapt to an ever-changing length. Little attention has been given to the effect of time on ASM shortening. The present study investigates the effect of changing the time interval between simulated deep inspirations (DIs) on ASM shortening and its responsiveness to simulated DIs. Excised tracheal strips from sheep were mounted in organ baths and either activated with methacholine or relaxed with isoproterenol. They were then subjected to simulated DIs by imposing swings in distending stress, emulating a transmural pressure from 5 to 30 cmH2O. The simulated DIs were intercalated by 2, 5, 10, or 30 min. In between simulated DIs, the distending stress was either fixed or oscillating to simulate tidal breathing. The results show that although shortening was increased by prolonging the interval between simulated DIs, the bronchodilator effect of simulated DIs (i.e., the elongation of the strip post- vs. pre-DI) was not affected, and the rate of re-shortening post-simulated DIs was decreased. As the frequency with which DIs are taken increases upon bronchoconstriction, our results may be relevant to typical alterations observed in asthma, such as an increased rate of re-narrowing post-DI