Infection with Influenzavirus A in a murine model induces epithelial bronchial lesions and distinct waves of innate immune-cell recruitment.

Introduction: Inflammatory lesions after Influenza A viruses (IAV) are potential therapeutic target for which better understanding of post-infection immune mechanisms is required. Most studies to evaluate innate immune reactions induced by IAV are based on quantitative/functional methods and anatomical exploration is most often non-existent. We aimed to study pulmonary damage and macrophage recruitment using two-photon excitation microscopy (TPEM) after IAV infection. Methods: We infected C57BL/6 CD11c+YFP mice with A/Puerto Ricco/8/34 H1N1. We performed immune cell analysis, including flow cytometry, cytokine concentration assays, and TPEM observations after staining with anti-F4/80 antibody coupled to BV421. We adapted live lung slice (LLS) method for ex-vivo intravital microscopy to analyze cell motility. Results: TPEM provided complementary data to flow cytometry and cytokine assays by allowing observation of bronchial epithelium lesions and spreading of local infection. Addition of F4/80-BV421 staining allowed us to precisely determine timing of recruitment and pulmonary migration of macrophages. Ex-vivo LLS preserved cellular viability, allowing us to observe acceleration of macrophage motility. Conclusion: After IAV infection, we were able to explore structural consequences and successive waves of innate immune cell recruitment. By combining microscopy, flow cytometry and chemokine measurements, we describe novel and precise scenario of innate immune response against IAV.

Long-term systemic and mucosal SARS-CoV-2 IgA response and its association with persistent smell and taste disorders.

Introduction: Current approved COVID-19 vaccines, notably mRNA and adenoviral vectored technologies, still fail to fully protect against infection and transmission of various SARS-CoV-2 variants. The mucosal immunity at the upper respiratory tract represents the first line of defense against respiratory viruses such as SARS-CoV-2 and is thus critical to develop vaccine blocking human-to-human transmission. Methods: We measured systemic and mucosal Immunoglobulin A (IgA) response in serum and saliva from 133 healthcare workers from Percy teaching military hospital following a mild infection (SARS-CoV-2 Wuhan strain, n=58) or not infected (n=75), and after SARS-CoV-2 vaccination (Vaxzevria®/Astrazeneca and/or Comirnaty®/Pfizer). Results: While serum anti-SARS-CoV-2 Spike IgA response lasted up to 16 months post-infection, IgA response in saliva had mostly fallen to baseline level at 6 months post-infection. Vaccination could reactivate the mucosal response generated by prior infection, but failed to induce a significant mucosal IgA response by itself. Early post-COVID-19 serum anti-Spike-NTD IgA titer correlated with seroneutralization titers. Interestingly, its saliva counterpart positively correlated with persistent smell and taste disorders more than one year after mild COVID-19. Discussion: As breakthrough infections have been correlated with IgA levels, other vaccine platforms inducing a better mucosal immunity are needed to control COVID-19 infection in the future. Our results encourage further studies to explore the prognosis potential of anti-Spike-NTD IgA in saliva at predicting persistent smell and taste disorders.

A live measles-vectored COVID-19 vaccine induces strong immunity and protection from SARS-CoV-2 challenge in mice and hamsters.

Several COVID-19 vaccines have now been deployed to tackle the SARS-CoV-2 pandemic, most of them based on messenger RNA or adenovirus vectors.The duration of protection afforded by these vaccines is unknown, as well as their capacity to protect from emerging new variants. To provide sufficient coverage for the world population, additional strategies need to be tested. The live pediatric measles vaccine (MV) is an attractive approach, given its extensive safety and efficacy history, along with its established large-scale manufacturing capacity. We develop an MV-based SARS-CoV-2 vaccine expressing the prefusion-stabilized, membrane-anchored full-length S antigen, which proves to be efficient at eliciting strong Th1-dominant T-cell responses and high neutralizing antibody titers. In both mouse and golden Syrian hamster models, these responses protect the animals from intranasal infectious challenge. Additionally, the elicited antibodies efficiently neutralize in vitro the three currently circulating variants of SARS-CoV-2.

Differential serological and neutralizing antibody dynamics after an infection by a single SARS-CoV-2 strain.

BACKGROUND: We report here the case of two coworkers infected by the same SARS-CoV-2 strain, presenting two different immunological outcomes. CASE: One patient presented a strong IgG anti-receptor-binding domain immune response correlated with a low and rapidly decreasing titer of neutralizing antibodies. The other patient had a similar strong IgG anti-receptor-binding domain immune response but high neutralizing antibody titers. DISCUSSION AND CONCLUSION: Thus, host individual factors may be the main drivers of the immune response varying with age and clinical severity.

[COVID-19 and vaccination: a global disruption]. / COVID-19 et vaccination : une dérégulation globale.

Coronavirus disease (COVID)-19 is an emerging pandemic infection whose significant ability to spread in a naïve population is well established. The first response of states to the COVID-19 outbreak was to impose lock-down and social barrier measures, such as wearing a surgical mask or social distancing. One of the consequences of this pandemic in terms of public health was the suspension or slowdown of infant vaccination campaigns, in almost all countries. The indirect effects of COVID-19 may therefore weigh on mortality from measles and polio in developing countries. In this pandemic chaos, the only hope lies in the rapid development of an effective vaccine against severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). However, acceptance of this vaccine has not yet been won, as beyond the many unknowns that will inevitably weigh around such rapid development, skepticism among vaccine hesitants is growing.

TITLE: COVID-19 et vaccination : une dérégulation globale. ABSTRACT: La COVID-19 est une infection pandémique émergente dont l'importante capacité à se propager dans une population dénuée d'immunité n'est plus à prouver. La première réponse des États à la flambée de COVID-19 fut d'imposer un confinement et des mesures barrières, telles que le port du masque et la distanciation sociale. Une des répercussions de cette pandémie, en matière de santé publique, fut la suspension ou le ralentissement brusque des campagnes de vaccination des nourrissons, un peu partout dans le monde. Un des effets indirects de la COVID-19 est donc le risque de peser sur la mortalité mondiale, principalement via une recrudescence de la rougeole et de la poliomyélite, principalement dans les pays en voie de développement. Dans ce chaos potentiel, le seul espoir réside dans le développement rapide d'un vaccin efficace contre le SARS-CoV-2 (severe acute respiratory syndrome-coronavirus-2). Cependant, l'acceptation de ce vaccin par la population n'est pas évidente, car outre les nombreuses inconnues qui vont peser inévitablement dans le cas d'un développement très rapide du vaccin, le scepticisme des hésitants vaccinaux va à nouveau se développer.

Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo.

This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a so-called fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, provides high labeling selectivity. Y-FAST was engineered from the 14-kDa photoactive yellow protein by directed evolution using yeast display and fluorescence-activated cell sorting. Y-FAST is as bright as common fluorescent proteins, exhibits good photostability, and allows the efficient labeling of proteins in various organelles and hosts. Upon fluorogen binding, fluorescence appears instantaneously, allowing monitoring of rapid processes in near real time. Y-FAST distinguishes itself from other tagging systems because the fluorogen binding is highly dynamic and fully reversible, which enables rapid labeling and unlabeling of proteins by addition and withdrawal of the fluorogen, opening new exciting prospects for the development of multiplexing imaging protocols based on sequential labeling.

Light-Activated Proteolysis for the Spatiotemporal Control of Proteins.

The regulation of proteolysis is an efficient way to control protein function in cells. Here, we present a general strategy enabling to increase the spatiotemporal resolution of conditional proteolysis by using light activation as trigger. Our approach relies on the auxin-inducible degradation system obtained by transposing components of the plant auxin-dependent degradation pathway in mammalian cells. We developed a photoactivatable auxin that acts as a photoactivatable inducer of degradation. Upon local and short light illumination, auxin is released in cells and triggers the degradation of a protein of interest with spatiotemporal control.

Inhibition of lipid phosphate phosphatase activity by VPC32183 suppresses the ability of diacylglycerol pyrophosphate to activate ERK(1/2) MAP kinases.

The lipidic metabolite, diacylglycerol pyrophosphate (DGPP), in its dioctanoyl form (DGPP 8:0), has been described as an antagonist for mammalian lysophosphatidic acid (LPA) receptors LPA1 and LPA3. In this study we show that DGPP 8:0 does not antagonize LPA dependent activation of ERK(1/2) MAP kinases but strongly stimulated them in various mammalian cell lines. LPA and DGPP 8:0 stimulation of ERK(1/2) occurred through different pathways. The DGPP 8:0 effect appeared to be dependent on PKC, Raf and MEK but was insensitive to pertussis toxin and did not involve G protein activation. Finally we showed that DGPP 8:0 effect on ERK(1/2) was dependent on its dephosphorylation by a phosphatase activity sharing lipid phosphate phosphatase properties. The inhibition of this phosphatase activity by VPC32183, a previously characterized LPA receptor antagonist, blocked the DGPP 8:0 effect on ERK(1/2) activation. Moreover, down-regulation of lipid phosphate phosphatase 1 (LPP1) expression by RNA interference technique also reduced DGPP 8:0-induced ERK(1/2) activation. Consistently, over expression of LPP1 in HEK293 cells increases DGPP 8:0 hydrolysis and this increased activity was inhibited by VPC32183. In conclusion, DGPP 8:0 does not exert its effect by acting on a G protein coupled receptor, but through its dephosphorylation by LPP1, generating dioctanoyl phosphatidic acid which in turn activates PKC. These results suggest that LPP1 could have a positive regulatory function on cellular signaling processes such as ERK(1/2) activation.

Non-ionic amphiphilic homopolymers: synthesis, solution properties, and biochemical validation.

A novel type of nonionic amphipols for handling membrane proteins in detergent-free aqueous solutions has been obtained through free-radical homo-telomerization of an acrylamide-based monomer comprising a C(11) alkyl chain and two glucose moieties, using a thiol as transfer reagent. By controlling the thiol/monomer ratio, the number-average molecular weight of the polymers was varied from 8 to 63 kDa. Homopolymeric nonionic amphipols were found to be highly soluble in water and to self-organize, within a large concentration range, into small, compact particles of ~6 nm diameter with a narrow size distribution, regardless of the molecular weight of the polymer. They proved able to trap and stabilize two test membrane proteins, bacteriorhodopsin from Halobium salinarum and the outer membrane protein X of Escherichia coli, under the form of small and well-defined complexes, whose size, composition, and shape were studied by aqueous size-exclusion chromatography, analytical ultracentrifugation, and small-angle neutron scattering. As shown in a companion paper, nonionic amphipols can be used for membrane protein folding, cell-free synthesis, and solution NMR studies (Bazzacco et al. 2012, Biochemistry, DOI: 10.1021/bi201862v).

Nonionic homopolymeric amphipols: application to membrane protein folding, cell-free synthesis, and solution nuclear magnetic resonance.

Nonionic amphipols (NAPols) synthesized by homotelomerization of an amphiphatic monomer are able to keep membrane proteins (MPs) stable and functional in the absence of detergent. Some of their biochemical and biophysical properties and applications have been examined, with particular attention being paid to their complementarity with the classical polyacrylate-based amphipol A8-35. Bacteriorhodopsin (BR) from Halobacterium salinarum and the cytochrome b(6)f complex from Chlamydomonas reinhardtii were found to be in their native state and highly stable following complexation with NAPols. NAPol-trapped BR was shown to undergo its complete photocycle. Because of the pH insensitivity of NAPols, solution nuclear magnetic resonance (NMR) two-dimensional (1)H-(15)N heteronuclear single-quantum coherence spectra of NAPol-trapped outer MP X from Escherichia coli (OmpX) could be recorded at pH 6.8. They present a resolution similar to that of the spectra of OmpX/A8-35 complexes recorded at pH 8.0 and give access to signals from solvent-exposed rapidy exchanging amide protons. Like A8-35, NAPols can be used to fold MPs to their native state as demonstrated here with BR and with the ghrelin G protein-coupled receptor GHS-R1a, thus extending the range of accessible folding conditions. Following NAPol-assisted folding, GHS-R1a bound four of its specific ligands, recruited arrestin-2, and activated binding of GTPγS by the G(αq) protein. Finally, cell-free synthesis of MPs, which is inhibited by A8-35 and sulfonated amphipols, was found to be very efficient in the presence of NAPols. These results open broad new perspectives on the use of amphipols for MP studies.

In the cauldron of cell-free synthesis of membrane proteins: playing with new surfactants.

Cell-free protein synthesis is a well-known technique for the roles it has played in deciphering the genetic code and in the beginnings of signal sequence studies. Since then, many efforts have been made to optimise this technique and, recently, to adapt it to membrane protein production with yields compatible with structural investigations. The versatility of the method allows membrane proteins to be obtained directly stabilised in surfactant micelles or inserted in a lipidic environment (proteoliposome, bicelle, and nanodisc) at the end of synthesis. Among the surfactants used, non-detergent ones such as fluorinated surfactants proved to be a good alternative in terms of colloidal stability and preservation of the integrity of membrane proteins, as shown for Escherichia coli homo-pentameric channel, MscL (Park et al., Biochem. J., 403: 183-187). Here we report cell-free expression of Escherichia coli leader peptidase (a transmembrane protease), Halobacterium salinarium bacteriorhodopsin (a transmembrane protein binding a hydrophobic cofactor) and E. coli MscL in the presence of non-detergent surfactants, amphipols and fluorinated surfactants in comparison to their expression in classical detergents. The results confirm the potentialities of fluorinated surfactants and, although pointing to limitations in using the first generations amphipols, results are discussed in the light of membrane protein refolding, especially in the case of bacteriorhodopsin. Preliminary experiments using new generations of amphipols supports choices made in developing new molecules.

Lysophosphatidic acid signaling during embryo development in sheep: involvement in prostaglandin synthesis.

We investigated the lysophosphatidic acid (LPA) pathway during early pregnancy in sheep. LPA was detected in the uteri of early-stage pregnant ewes. Using quantitative RT-PCR, the expression of autotaxin, the LPA-generating enzyme, was found in the endometrium and conceptus. In the latter autotaxin, transcript levels were low on d 12-14 and increased on d 15-16, in parallel with the level of LPA. Autotaxin was localized in the luminal epithelium and superficial glands of the endometrium and in trophectoderm cells of the conceptus. The expression of G protein-coupled receptors for LPA was also examined in the ovine conceptus. LPA receptor LPAR1 and LPAR3 transcripts were expressed during early pregnancy and displayed a peak on d 14, whereas the highest level of protein for both receptors was observed at d 17. LPAR1 was localized in cellular membranes and nuclear compartments of the trophectoderm cells, whereas LPAR3 was revealed only in membranes. LPA activated phosphorylation of the MAPK ERK1/2 in ovine trophectoderm-derived cells. Moreover, the bioactive lipid increased the proliferation of trophectoderm cells in culture, as shown by thymidine and bromodeoxyuridine incorporation. Furthermore, LPA induced changes to the organization of beta-actin and alpha-tubulin, suggesting a role for it in rearrangement of trophectoderm cells cytoskeleton. Because a link had previously been established between prostaglandin and LPA pathways, we analyzed the effect of LPA on prostaglandin synthesis. LPA induced an increase in the release of prostaglandin F2alpha and prostaglandin E2, with no significant modifications to cytosolic phospholipase A2alpha and prostaglandin synthase-2 expression. Taken together, our results suggest a new role for LPA-mediated signaling in the ovine conceptus at the time of implantation.

Role of lysophosphatidic acid in the regulation of uterine leiomyoma cell proliferation by phospholipase D and autotaxin.

Phospholipase D (PLD) hydrolyzes phosphatidylcholine into phosphatidic acid (PA), a lipidic mediator that may act directly on cellular proteins or may be metabolized into lysophosphatidic acid (LPA). We previously showed that PLD contributed to the mitogenic effect of endothelin-1 (ET-1) in a leiomyoma cell line (ELT3 cells). In this work, we tested the ability of exogenous PA and PLD from Streptomyces chromofuscus (scPLD) to reproduce the effect of endogenous PLD in ELT3 cells and the possibility that these agents acted through LPA formation. We found that PA, scPLD, and LPA stimulated thymidine incorporation. LPA and scPLD induced extracellular signal-regulated kinase (ERK(1/2)) mitogen-activated protein kinase activation. Using Ki16425, an LPA(1)/LPA(3) receptor antagonist and small interfering RNA targeting LPA(1) receptor, we demonstrated that scPLD acted through LPA production and LPA(1) receptor activation. We found that scPLD induced LPA production by hydrolyzing lysophosphatidylcholine through its lysophospholipase D (lysoPLD) activity. Autotaxin (ATX), a naturally occurring lysoPLD, reproduced the effects of scPLD. By contrast, endogenous PLD stimulated by ET-1 failed to produce LPA. These results demonstrate that scPLD stimulated ELT3 cell proliferation by an LPA-dependent mechanism, different from that triggered by endogenous PLD. These data suggest that in vivo, an extracellular lysoPLD such as ATX may participate in leiomyoma growth through local LPA formation.

Direct interaction of surfactant protein A with myometrial binding sites: signaling and modulation by bacterial lipopolysaccharide.

Surfactant protein A (SFTPA1), a member of the collagenous lectin (collectin) family, was first described as a major constituent of lung surfactant, but has recently also been found in the female genital tract. Various microorganisms colonize this area and may cause intrauterine infection or trigger preterm labor. We found that SFTPA1 was not produced in the uterus. Instead, it was immunodetected transiently in rat myometrium at the end (Days 19 and 21) of gestation, but not postpartum, and in cultured myometrial cells. Fluorescence microscopy showed that Texas Red-labeled SFTPA1 bound to myometrial cells. This result was confirmed by biochemical approaches. [(125)I]-SFTPA1 bound to two myometrial cell proteins (55 and 210 kDa). This interaction was dependent on the integrity of the collagenlike domain of SFTPA1. SFTPA1 rapidly activated mitogen-activated protein kinase 1/3 (MAPK1/3) in myometrial cells. Bacterial lipopolysaccharide (LPS), an agent known to trigger uterine contractions and preterm birth, also activated MAPK1/3. The prolonged treatment of myometrial cells with LPS or SFTPA1 upregulated PTGS2 (COX2) protein levels. The addition of rough-type LPS to SFTPA1 blocked the interaction of SFTPA1 with its binding sites and the activation of MAPK1/3 and PTGS2 by SFTPA1. Our data provide the first demonstration of a direct effect of SFTPA1 on rat myometrial cells and inhibitory cross talk between SFTPA1 and LPS signals, providing new insight into the mechanisms of normal and preterm parturition.