Interleukin-23-Independent IL-17 Production Regulates Intestinal Epithelial Permeability

SummaryWhether interleukin-17A (IL-17A) has pathogenic and/or protective roles in the gut mucosa is controversial and few studies have analyzed specific cell populations for protective functions within the inflamed colonic tissue. Here we have provided evidence for IL-17A-dependent regulation of the tight junction protein occludin during epithelial injury that limits excessive permeability and maintains barrier integrity. Analysis of epithelial cells showed that in the absence of signaling via the IL-17 receptor adaptor protein Act-1, the protective effect of IL-17A was abrogated and inflammation was enhanced. We have demonstrated that after acute intestinal injury, IL-23R+ γδ T cells in the colonic lamina propria were the primary producers of early, gut-protective IL-17A, and this production of IL-17A was IL-23 independent, leaving protective IL-17 intact in the absence of IL-23. These results suggest that IL-17-producing γδ T cells are important for the maintenance and protection of epithelial barriers in the intestinal mucosa

Fc-mediated functions of nirsevimab complement direct respiratory syncytial virus neutralization but are not required for optimal prophylactic protection

IntroductionNirsevimab is an extended half-life (M252Y/S254T/T256E [YTE]-modified) monoclonal antibody to the pre-fusion conformation of the respiratory syncytial virus (RSV) Fusion protein, with established efficacy in preventing RSV-associated lower respiratory tract infection in infants for the duration of a typical RSV season. Previous studies suggest that nirsevimab confers protection via direct virus neutralization. Here we use preclinical models to explore whether fragment crystallizable (Fc)-mediated effector functions contribute to nirsevimab-mediated protection.MethodsNirsevimab, MEDI8897* (i.e., nirsevimab without the YTE modification), and MEDI8897*-TM (i.e., MEDI8897* without Fc effector functions) binding to Fc γ receptors (FcγRs) was evaluated using surface plasmon resonance. Antibody-dependent neutrophil phagocytosis (ADNP), antibody-dependent cellular phagocytosis (ADCP), antibody-dependent complement deposition (ADCD), and antibody-dependent cellular cytotoxicity (ADCC) were assessed through in vitro and ex vivo serological analyses. A cotton rat challenge study was performed with MEDI8897* and MEDI8897*-TM to explore whether Fc effector functions contribute to protection from RSV.ResultsNirsevimab and MEDI8897* exhibited binding to a range of FcγRs, with expected reductions in FcγR binding affinities observed for MEDI8897*-TM. Nirsevimab exhibited in vitro ADNP, ADCP, ADCD, and ADCC activity above background levels, and similar ADNP, ADCP, and ADCD activity to palivizumab. Nirsevimab administration increased ex vivo ADNP, ADCP, and ADCD activity in participant serum from the MELODY study (NCT03979313). However, ADCC levels remained similar between nirsevimab and placebo. MEDI8897* and MEDI8897*-TM exhibited similar dose-dependent reduction in lung and nasal turbinate RSV titers in the cotton rat model.ConclusionNirsevimab possesses Fc effector activity comparable with the current standard of care, palivizumab. However, despite possessing the capacity for Fc effector activity, data from RSV challenge experiments illustrate that nirsevimab-mediated protection is primarily dependent on direct virus neutralization

Effets des antiandrogènes sur la prostate de rat (approches protéomiques et génomiques)

Les perturbateurs endocriniens sont des agents exogènes qui interfèrent avec les hormones naturelles agissant sur l'homéostasie, la reproduction, le développement ou le comportement des organismes. Leur abondance dans l'environnement pose un véritable problème de santé publique qui nécessite aujourd'hui de développer de nouvelles stratégies de recherche en toxicologie. Parmi celles-ci, la toxico-protéomique et la toxico-génomique sont en plein essor. La prostate, dont le développement et l'homéostasie sont sous le contrôle direct des androgènes, constitue un organe de choix pour étudier les effets des antiandrogènes, l'une des principales classes de perturbateurs endocriniens. Au cours de ce travail de thèse, nous avons ainsi étudié les effets de deux molécules de référence sur la prostate: le finastéride (inhibiteur de la 5 a-réductase de type 2), le flutamide (inhibiteur compétitif du récepteur aux androgènes). L'analyse toxicologique classique, définie par la directive 407 de l'OCDE étudie les effets sur un organisme engendrés par une exposition chronique de 28 jours à une substance. Elle comporte les mesures du poids des organes reproducteurs, des concentrations hormonales et des études histologiques. Ces analyses nous ont permis de confirmer l'activité antiandrogénique de ces 2 molécules, principalement caractérisée par une atrophie prostatique et une augmentation des concentrations circulantes de testostérone et d'hormone lutéinisante. L'étude comparative du protéome des prostates ventrales des animaux contrôles et traités pendant 28 jours nous a permis d'identifier 37 et 21 protéines, dont le niveau d'expression est modulé par l'exposition respectivement au finastéride et au flutamide. De manière intéressante, la fonction de ces protéines reflète l'atteinte du tissu épithélial prostatique : une diminution de l'activité sécrétoire, une dérégulation du stress oxydatif, de la détoxification et de l'apoptose, qui seraient responsables en partie de l'atrophie prostatique observée en réponse à la déprivation en androgènes. De plus, 7 protéines sont modulées de façon identique par les 2 antiandrogènes. Parmi celle-ci, nous avons mis en évidence, pour la première fois chez le rat, une protéine similaire à la L-amino acide oxydase-1 (Lao1) présente uniquement dans l'épithélium prostatique. Cette enzyme, dont l'expression est fortement augmentée par le finastéride et le flutamide, produit du peroxyde d'hydrogène qui pourrait jouer un rôle important dans l'atrophie prostatique induite par les antiandrogènes. Par ailleurs, une étude toxicologique aigüe a montré que le niveau d'expression de la Lao1 augmente fortement dès 48h après l'administration d'un bolus unique de flutamide, alors qu'aucune lésion tissulaire n'est observable histologiquement, ce qui fait de cette protéine un marqueur potentiel prédictif intéressant des effets des antiandrogènes. Enfin, l'étude du transcriptome des prostates ventrales de rats de cette dernière étude toxicologique aigüe, a révélé que la modulation d'expression de gènes impliqués dans la croissance, la prolifération et la mort cellulaire, le métabolisme des lipides et l'inflammation, déjà décrite après castration, intervient dès 8 h après traitement et constitue un marqueur prédictif précoce d'une activité antiandrogénique.NICE-BU Sciences (060882101) / SudocSudocFranceF

Inferior immunogenicity and efficacy of respiratory syncytial virus fusion protein-based subunit vaccine candidates in aged versus young mice.

Respiratory syncytial virus (RSV) is recognized as an important cause of lower and upper respiratory tract infections in older adults, and a successful vaccine would substantially lower morbidity and mortality in this age group. Recently, two vaccine candidates based on soluble purified glycoprotein F (RSV F), either alone or adjuvanted with glucopyranosyl lipid A formulated in a stable emulsion (GLA-SE), failed to reach their primary endpoints in clinical efficacy studies, despite demonstrating the desired immunogenicity profile and efficacy in young rodent models. Here, one of the RSV F vaccine candidates (post-fusion conformation, RSV post-F), and a stabilized pre-fusion form of RSV F (RSV pre-F, DS-Cav1) were evaluated in aged BALB/c mice. Humoral and cellular immunogenicity elicited after immunization of naïve, aged mice was generally lower compared to young animals. In aged mice, RSV post-F vaccination without adjuvant poorly protected the respiratory tract from virus replication, and addition of GLA-SE only improved protection in the lungs, but not in nasal turbinates. RSV pre-F induced higher neutralizing antibody titers compared to RSV post-F (as previously reported) but interestingly, RSV F-specific CD8 T cell responses were lower compared to RSV post-F responses regardless of age. The vaccines were also tested in RSV seropositive aged mice, in which both antigen forms similarly boosted neutralizing antibody titers, although GLA-SE addition boosted neutralizing activity only in RSV pre-F immunized animals. Cell-mediated immune responses in the aged mice were only slightly boosted and well below levels induced in seronegative young mice. Taken together, the findings suggest that the vaccine candidates were not able to induce a strong anti-RSV immune response in recipient mice with an aged immune system, in agreement with recent human clinical trial results. Therefore, the aged mouse model could be a useful tool to evaluate improved vaccine candidates, targeted to prevent RSV disease in older adults

Lung T cell profile after RSV challenge in young and aged seronegative mice immunized with post-RSV F formulations.

<p>Young (7 weeks old) and aged (18 months old) BALB/c mice (n = 4 to 7 per group) were immunized i.m. at day 0 and day 21 with buffer alone, post-F (0.3 μg) +/- GLA-SE (2.5 μg/2%) adjuvant. A control group for natural infection was immunized i.n. with live RSV (10⁶ PFU) at day 0. At day 35, animals were challenged i.n. with 10⁶ PFU of wt RSV A2. 4 days post-challenge (day 39), lung cells were isolated and stimulated with RSV F peptide pool to evaluate intracellular cytokine expression by flow cytometry. Cells were surface stained for CD3 and CD8, intracellularly stained for IFNγ, TNFα, and IL-2, and analyzed on an LSR II for the frequency of responding (A) CD4+ and (B) CD8+ T cells. (C) The percentage of F85-93-pentamer+ CD8 T cells was determined by flow cytometry. (D) The percentage of lung eosinophils was determined by flow cytometry. For (C) and (D), the mean of individual values +/- SD is shown. For statistical analyses, aged and young mice were compared. *, P<0.05.</p

Immunogenicity of post-F and pre-F in aged seronegative mice.

<p>Aged (18 months old) BALB/c mice (n = 6 to 10 per group) were immunized i.m. at day 0 and day 21 with buffer alone, RSV F (pre- or post- conformation) at the indicated doses +/- GLA-SE (2.5 μg/2%) adjuvant. A control group for natural infection was immunized i.n. with live RSV (10⁶ PFU) at day 0. At day 35, animals were challenged i.n. with 10⁶ PFU of wt RSV A2. Prior to challenge (day 34), sera were harvested and NAb titers were evaluated using a microneutralization assay. Data is presented as the log₂ dilution of serum that provides 50% reduction in viral entry with a LLOD of 4 indicated by a dashed line. 4 days post-RSV A2 challenge, splenocytes were isolated and stimulated for 24 h with peptides representing <b>(B)</b> RSV F specific CD4 T cell and <b>(C)</b> CD8 T cell epitopes. The number of IFNγ secreting cells per 10⁶ splenocytes was determined by ELISPOT. Group means ± SD of individual mice are shown. For statistical analyses, aged and young mice were compared. *, P<0.05.</p

Immunogenicity and protection from viral challenge of pre-RSV + GLA-SE in young, seronegative mice.

<p>Young (7 weeks old) BALB/c mice (n = 5 to 10 per group) were immunized i.m. at day 0 and day 21 with buffer alone, RSV F pre- and post-conformation at 0.3 μg +/- GLA-SE (2.5 μg/2%) adjuvant. At day 35, animals were challenged i.n. with 10⁶ PFU of wt RSV A2. (A) Prior to challenge (day 34), sera were harvested and NAb titers were evaluated using a microneutralization assay. Data is presented as the log₂ dilution of serum that provides 50% reduction in viral entry with a LLOD of 4 indicated by a dashed line. (B) 4 days post-challenge (day 39), splenocytes were isolated and stimulated for 24 h with RSV F specific CD4 T-cell and CD8 T cell epitopes. The number of IFNγ secreting cells per 10⁶ splenocytes was determined by ELISPOT. (C) Lung and (D) nasal turbinates viral loads were measured by plaque assay. Group means ± SD of individual mice are shown. For statistical analyses, aged and young mice were compared. *, P<0.05.</p

NAb induction of pre-F and post-F formulations in aged seropositive mice.

<p>12 months old mice were i.n. immunized with RSV A2 (10⁶ PFU) at day 0 and RSV B (10⁵ PFU) at day 60. Aged seropositive mice were then immunized i.m. at day 176 (18 months old) with buffer alone, post-F or pre-F (0.3 μg) +/- GLA-SE (2.5 μg/2%) adjuvant. <b>(A)</b> Bleeds were collected at day 50, 120, and 174 to assess the levels of RSV A2 neutralizing antibodies in seropositive animals prior to immunization (baseline). Data is presented as the log₂ dilution of serum that provides 50% reduction in viral entry with a LLOD of 4 indicated by a dashed line. <b>(B)</b> 14 days post immunization, RSV A2 neutralization titers were evaluated. Group means ± SD of individual mice are shown. For statistical analyses, multiplicity adjusted ANOVA test was performed against baseline as a control group, followed by pairwise testing of post-F versus pre-F, * for P<0.05.</p