[Cell senescence, a new target for respiratory viral infections: From influenza virus to SARS-CoV-2]. / La sénescence cellulaire, nouvelle cible des infections virales respiratoires : du virus influenza au SARS-CoV-2.

The accumulation of senescent cells in tissues is a key process of aging and age-related diseases, including lung diseases such as chronic obstructive pulmonary disease, lung fibrosis, or cancer. In recent years, the spectrum of respiratory diseases associated with cellular senescence has been broadened, in particular acute viral pulmonary infections, foremost among which is coronavirus disease 2019 (COVID19), which is particularly severe in the elderly or in subjects with comorbidities. Influenza virus infection, which strikes more severely at the extreme ages of life, is also associated with severe pulmonary senescence. Cellular senescence potentially represents an original target for attacking these diseases, although its specific mechanisms remain largely misunderstood. New anti-senescent therapeutic approaches are thus proposed during severe viral pulmonary infections, with the aim of preventing acute effects and/or, in the longer term, pulmonary sequelae.

Boosting the IL-22 response using flagellin prevents bacterial infection in cigarette smoke-exposed mice.

The progression of chronic obstructive pulmonary disease (COPD), a lung inflammatory disease being the fourth cause of death worldwide, is marked by acute exacerbations. These episodes are mainly caused by bacterial infections, frequently due to Streptococcus pneumoniae. This susceptibility to infection involves a defect in interleukin (IL)-22, which plays a pivotal role in mucosal defense mechanism. Administration of flagellin, a Toll-like receptor 5 (TLR-5) agonist, can protect mice and primates against respiratory infections in a non-pathological background. We hypothesized that TLR-5-mediated stimulation of innate immunity might improve the development of bacteria-induced exacerbations in a COPD context. Mice chronically exposed to cigarette smoke (CS), mimicking COPD symptoms, are infected with S. pneumoniae, and treated in a preventive and a delayed manner with flagellin. Both treatments induced a lower bacterial load in the lungs and blood, and strongly reduced the inflammation and lung lesions associated with the infection. This protection implicated an enhanced production of IL-22 and involved the recirculation of soluble factors secreted by spleen cells. This is also associated with higher levels of the S100A8 anti-microbial peptide in the lung. Furthermore, human mononuclear cells from non-smokers were able to respond to recombinant flagellin by increasing IL-22 production while active smoker cells do not, a defect associated with an altered IL-23 production. This study shows that stimulation of innate immunity by a TLR-5 ligand reduces CS-induced susceptibility to bacterial infection in mice, and should be considered in therapeutic strategies against COPD exacerbations.

CD3bright signals on γδ T cells identify IL-17A-producing Vγ6Vδ1+ T cells.

Interleukin-17A (IL-17A) is a pro-inflammatory cytokine that has an important role at mucosal sites in a wide range of immune responses including infection, allergy and auto-immunity. γδ T cells are recognized as IL-17 producers, but based on the level of CD3 expression, we now define the remarkable ability of a CD3(bright) γδ T-cell subset with an effector memory phenotype to rapidly produce IL-17A, but not interferon-γ. CD3(bright) γδ T cells uniformly express the canonical germline encoded Vγ6/Vδ1(+) T-cell receptor. They are widely distributed with a preferential representation in the lungs and skin are negatively impacted in the absence of retinoic acid receptor-related orphan receptor gammat expression or endogenous flora. This population responded rapidly to various stimuli in a mechanism involving IL-23 and NOD-like receptor family, pyrin domain containing 3 (NLRP3)-inflammasome-dependent IL-1ß. Finally, we demonstrated that IL-17-producing CD3(bright) γδ T cells responded promptly and strongly to pneumococcal infection and during skin inflammation. Here, we propose a new way to specifically analyze IL-17-producing Vγ6/Vδ1(+) T cells based on the level of CD3 signals. Using this gating strategy, our data reinforce the crucial role of this γδ T-cell subset in respiratory and skin disorders.

Role of the parasite-derived prostaglandin D2 in the inhibition of epidermal Langerhans cell migration during schistosomiasis infection.

Epidermal Langerhans cells (LCs) play a key role in immune defense mechanisms and in numerous immunological disorders. In this report, we show that percutaneous infection of C57BL/6 mice with the helminth parasite Schistosoma mansoni leads to the activation of LCs but, surprisingly, to their retention in the epidermis. Moreover, using an experimental model of LC migration induced by tumor necrosis factor (TNF)-alpha, we show that parasites transiently impair the departure of LCs from the epidermis and their subsequent accumulation as dendritic cells in the draining lymph nodes. The inhibitory effect is mediated by soluble lipophilic factors released by the parasites and not by host-derived antiinflammatory cytokines, such as interleukin-10. We find that prostaglandin (PG)D2, but not the other major eicosanoids produced by the parasites, specifically impedes the TNF-alpha-triggered migration of LCs through the adenylate cyclase-coupled PGD2 receptor (DP receptor). Moreover, the potent DP receptor antagonist BW A868C restores LC migration in infected mice. Finally, in a model of contact allergen-induced LC migration, we show that activation of the DP receptor not only inhibits LC emigration but also dramatically reduces the contact hypersensitivity responses after challenge. Taken together, we propose that the inhibition of LC migration could represent an additional stratagem for the schistosomes to escape the host immune system and that PGD2 may play a key role in the control of cutaneous immune responses.

Role of natural killer T lymphocytes during helminthic infection.

Natural killer (NK)T cells are innate lymphocytes that release important amount of immunoregulatory cytokines (IFN-gamma and/or IL-4) shortly after T cell receptor engagement by (glyco)lipid antigens presented by the CD1d molecules. Through this property, NKT cells play pivotal role in many physiopathologic situations. Here, we review the current knowledge of the functions and mechanisms of activation of NKT cells during infection, with a particular emphasis on helminthic infections. Recent findings suggest that, although dispensable for host resistance, NKT cells play part in the development of the acquired immune response and in the control of the pathology during murine schistosomiasis.

Influenza A virus-induced release of interleukin-10 inhibits the anti-microbial activities of invariant natural killer T cells during invasive pneumococcal superinfection.

During influenza A virus (IAV) infection, changes in the lung's physical and immunological defenses predispose the host to bacterial superinfections. Invariant natural killer T (iNKT) cells are innate-like T lymphocytes that have beneficial or harmful functions during infection. We investigated the iNKT cells' role in a model of invasive pneumococcal superinfection. The use of Jα18-/- mice indicated that iNKT cells limited susceptibility to influenza-pneumococcal infection and reduced the lethal synergism. This role did not depend on immune-based anti-bacterial mechanisms. At the time of bacterial exposure, iNKT cells from IAV-experienced mice failed to produce antipneumococcal interferon-γ and adoptive transfer of fresh iNKT cells before Streptococcus pneumoniae challenge did not restore anti-bacterial host defenses. Impaired iNKT cell activation in superinfected animals was related to the IAV-induced immunosuppressive cytokine interleukin-10 (IL-10), rather than to an intrinsic functional defect. IL-10 dampened the activation of iNKT cells in response to pneumococci by inhibiting the production of IL-12 by pulmonary monocyte-derived dendritic cells. Neutralization of IL-10 restored iNKT cell activation and tends to increase resistance to secondary bacterial infection. Overall, iNKT cells have a beneficial role (upstream of bacterial colonization) in controlling influenza-pneumococcal superinfection, although they represent novel targets of immunosuppression at the time of bacterial challenge.

Interleukin-22 protects against non-typeable Haemophilus influenzae infection: alteration during chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease is a major health problem becoming a leading cause of morbidity and mortality worldwide. A large part of these disorders is associated with acute exacerbations resulting from infection by bacteria, such as non-typeable Haemophilus influenzae (NTHi). Our understanding of the pathogenesis of these exacerbations is still elusive. We demonstrate herein that NTHi infection of mice chronically exposed to cigarette smoke (CS), an experimental model of chronic obstructive pulmonary disease (COPD), not only causes acute pulmonary inflammation but also impairs the production of interleukin (IL)-22, a cytokine with potential anti-bacterial activities. We also report that mice lacking IL-22, as well as mice exposed to CS, have a delayed clearance of NTHi bacteria and display enhanced alveolar wall thickening and airway remodeling compared with controls. Supplementation with IL-22 not only boosted bacterial clearance and the production of anti-microbial peptides but also limited lung damages induced by infection both in IL-22-/- and CS-exposed mice. In vitro exposure to CS extract altered the NTHi-induced IL-22 production by spleen cells. This study shows for the first time that a defect in IL-22 is involved in the acute exacerbation induced by NTHi infection during experimental COPD and opens the way to innovative therapeutic strategies.

Neutrophilic NLRP3 inflammasome-dependent IL-1ß secretion regulates the γδT17 cell response in respiratory bacterial infections.

Traditionally regarded as simple foot soldiers of the innate immune response limited to the eradication of pathogens, neutrophils recently emerged as more complex cells endowed with a set of immunoregulatory functions. Using a model of invasive pneumococcal disease, we highlighted an unexpected key role for neutrophils as accessory cells in innate interleukin (IL)-17A production by lung resident Vγ6Vδ1+ T cells via nucleotide-binding oligomerization domain receptor, pyrin-containing 3 (NLRP3) inflammasome-dependent IL-1ß secretion. In vivo activation of the NLRP3 inflammasome in neutrophils required both host-derived and bacterial-derived signals. Elaborately, it relies on (i) alveolar macrophage-secreted TNF-α for priming and (ii) subsequent exposure to bacterial pneumolysin for activation. Interestingly, this mechanism can be translated to human neutrophils. Our work revealed the cellular and molecular dynamic events leading to γδT17 cell activation, and highlighted for the first time the existence of a fully functional NLRP3 inflammasome in lung neutrophils. This immune axis thus regulates the development of a protective host response to respiratory bacterial infections.

Crystallization and preliminary X-ray diffraction studies of a protective cloned 28 kDa glutathione S-transferase from Schistosoma mansoni.

Crystals of the recombinant 28 kDa glutathione S-transferase from Schistosoma mansoni have been obtained by the hanging-drop method of vapor diffusion from ammonium sulfate solutions. The successful crystallization of this enzyme required the presence of a reducing agent and S-hexylglutathione. The crystals belong to the cubic space group P4(1)32 (or P4(3)32), with unit cell dimensions a = 122.6 A and contain one molecule in the asymmetric unit. The crystals diffract to at least 2.8 A resolution and are suitable for X-ray crystallographic structure analysis.

Cloning of a new cation ATPase from Plasmodium falciparum: conservation of critical amino acids involved in calcium binding in mammalian organellar Ca(2+)-ATPases.

In order to study molecules that may be involved in pH gradient formation in Plasmodium, we have identified a novel cation-translocating ATPase (P-type ATPase) gene from P. falciparum (Pf). We report the full-length nucleotide and deduced amino acid (aa) sequences of this gene that we called PfATPase4. The PfATPase4 protein shares features with the different members of eukaryotic P-type ATPases, such as a similar transmembrane (TM) organization and aa identity in functionally important regions. Interestingly, the PfATPase4 protein possesses conserved aa involved in calcium binding in mammalian organellar Ca(2+)-ATPases.

Peroxisome proliferator-activated receptor gamma activators inhibit interleukin-12 production in murine dendritic cells.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor superfamily. They are divided into three subtypes (alpha, beta or delta, and gamma) and are involved in lipid and glucose homeostasis and in the control of inflammation. In this study, we analyzed the expression of PPARs in murine dendritic cells (DCs), the most potent antigen presenting cells. We find that immature as well as mature spleen-derived DCs express PPARgamma, but not PPARalpha, mRNA and protein. We also show that the PPARgamma activator rosiglitazone does not interfere with the maturation of DCs in vitro nor modifies their ability to activate naive T lymphocytes in vivo. Finally, we present evidence that PPARgamma activators down-modulate the CD40-induced secretion of interleukin-12, a potent Th1-driving factor. These data suggest a possible role for PPARgamma in the regulation of immune responses.

Molecular cloning of a gene from Plasmodium falciparum that codes for a protein sharing motifs found in adhesive molecules from mammals and plasmodia.

Adhesion of Plasmodium to host cells is an important phenomenon in parasite invasion and in malaria-associated pathology. We report here the molecular cloning of a putative adhesive molecule from P. falciparum that shares both sequence and structural similarities with a sporozoite surface molecule from Plasmodium termed the thrombospondin-related anonymous protein (TRAP) and, to a lesser extent, with the circumsporozoite (CS) protein. The gene, which is present on chromosome 3 as a single copy, was termed CTRP for CS protein-TRAP-related protein. The full-length CTRP encodes a protein containing a putative signal sequence followed by a long extracellular region of 1990 amino acids, a transmembrane domain, and a short cytoplasmic segment. The putative extracellular region of CTRP is defined by two separated adhesive domains. The first domain contains six 210-amino acid-long homologous repeats, the sequence of which is related to the A-type domain found in adhesive molecules including the alpha subunits of several integrins and a number of extracellular matrix glycoproteins. The second domain contains seven repeats of 87-60 amino acids in length, which share similarities with the thrombospondin type 1 domain found in a variety of adhesive molecules. Finally, CTRP also contains consensus motifs found in the superfamily of haematopoietin receptors. Interstrain analysis of eight different parasite isolates revealed that CTRP does not show size polymorphism except in repetitive regions flanking potential adhesive domains.

Cloning and characterization of the vacuolar ATPase B subunit from Plasmodium falciparum.

The transvacuolar pH gradient determines, to a significant extent, the distribution of the antimalarial drug chloroquine in Plasmodium falciparum. A proton pump, similar to the vacuolar ATPase found in many cell types, appears to regulate a pH gradient across the membranes of acidic compartments of the parasite. In order to understand and define the components involved in the maintenance of the vacuolar pH gradient, we have cloned and characterized a gene, designated VAP B, encoding a P. falciparum homologue of the B subunit of the vacuolar ATPase. The VAP B gene encodes a protein of 494 amino acids which has between 69% and 74% amino acid identity with the sequences of vacuolar ATPase B subunits of other organisms. The VAP B gene exists as a single copy gene on chromosome 4 that gives rise to a RNA transcript of 2.4 kb. Antibodies raised to the VAP B protein react specifically with a protein of 56-kDa, consistent with the size predicted from the gene sequence and with the homologous protein from other organisms. The 56-kDa protein is expressed throughout the asexual life cycle and subcellular localization by indirect immunofluorescence shows that the protein has a heterogeneous distribution over most of the parasite. This suggests that the function of the vacuolar proton ATPase is not confined to the regulation of the pH of the digestive vacuole.

Cloning of the gene encoding a Schistosoma mansoni antigen homologous to human Ro/SS-A autoantigen.

A cDNA library was constructed from the mRNA of adult worms of Schistomsoma mansoni in the expression vector lambda gt11 and screened with a rabbit antiserum raised against a 60-65-kDa electroeluted adult worm fraction. Two overlapping clones were selected and a partial nucleotide sequence was deduced (1172 bp). The full-length sequence was obtained by the amplification of the 5' end of first strand cDNA using PCR. The overall mRNA size was 1335 nt including a 25 nt 5' non-coding region and a 131 nt untranslated region with the poly(A) tail. The predicted amino acid sequence of 393 aa (45 kDa) has 52% identity with the human Ro/SS-A autoantigen, which is considered to be the human calreticulin. As for the human Ro/SS-A, the protein encoded by the cDNA described here contains a hydrophobic leader sequence and a carboxyl terminal sequence, HDEL consensus signal sequence for retention in the ER. An antiserum raised against the fusion protein of one clone recognized a 58-kDa antigen in homogenates of cercariae and of adult worms. The expression of the protein in the pGEX-2T fusion system allowed us to show the presence of specific antibodies in S. mansoni infected patients' sera and in the sera of patients with systemic lupus erythematosus, reflecting a cross-immunoreactivity between the S. mansoni protein and the human calreticulin autoantigen.

Molecular cloning of a putative alpha3-fucosyltransferase from Schistosoma mansoni.

Alpha 3-fucosylation of protein or lipid substrates is an important component of the host/parasite interactions during schistosomiasis. In this process, alpha3-fucosyltransferases (alpha3-FucTs) are considered as key enzymes ensuring both parasite survival and adaptation in their (in)vertebrate hosts. In this paper, we report the molecular cloning of a putative alpha3-FucT from Schistosoma mansoni that we termed SmFucTA. The full-length SmFucTA encodes a typical transmembrane type II protein with a short cytoplasmic domain, a transmembrane segment and a long C-terminal catalytic domain. In this region, the GDP-fucose binding site is well conserved whereas the putative acceptor site displays sequence divergence compared to the corresponding region from vertebrate and invertebrate alpha3-FucTs. Southern blot analysis suggested that SmFucTA is present as several copies or has highly related counterparts in the S. mansoni genome. Northern blot revealed a single SmFucTA transcript at 2 kb in adult worms. Affinity purified antibodies directed against recombinant SmFucTA identified a 50 kDa native protein that localizes to the subtegumental and parenchymal regions of adult worms.

Inter-species variation of schistosome 28-kDa glutathione S-transferases.

The 28-kDa glutathione S-transferase from Schistosoma mansoni (Sm28GST) is a candidate vaccine antigen. To evaluate the antigenic and phylogenetic variations between the 28-kDa GSTs from 4 species of schistosome, we have cloned and sequenced the 28-kDa GSTs from Schistosoma haematobium (Sh28GST) and Schistosoma bovis (Sb28GST). Sb28GST and Sh28GST are more similar to each other (97%) than to Sm28GST (90%) and particularly to the 28-kDa GST from Schistosoma japonicum (Sj28GST, 77%). Antisera directed against the major Sm28GST epitopes revealed differences in the recognition of the 28-kDa GSTs from the other schistosome species suggesting that these regions have been subjected to evolutionary pressure. The consequences of such species-specific epitopes on the development of a multi-species anti-schistosome vaccine are discussed.

Molecular cloning and tissue distribution of a 26-kilodalton Schistosoma mansoni glutathione S-transferase.

A Schistosoma mansoni cDNA library was constructed from the mRNA of adult worms in the expression vector lambda gt11 and screened with a rabbit antiserum raised against the 26-kDa S. mansoni glutathione S-transferase isoforms (Sm GST 26). Two clones were selected and the nucleotide sequences deduced. The predicted amino acid sequence, specified by these cDNAs, shows strong homology with a Schistosoma japonicum 26 kDa glutathione S-transferase and a lower level of homology with mammalian glutathione S-transferase class mu isoenzymes (EC 2.5.1.18). No significant homology score was found with a 28-kDa S. mansoni glutathione S-transferase (Sm GST 28). A study of the tissue distribution of the cloned Sm GST 26 by immunoelectron microscopy shows similarities to Sm GST 28 in that they are present in the tegument and in subtegumentary parenchymal cells. However, a major difference exists in the protonephridial region in which Sm GST 26 is present in the cytoplasmic digitations localized in the apical chamber delineated by the flame cell body, suggesting that Sm GST 26 may be actively excreted by adult worms.

Isotypic analysis, antigen specificity, and inhibitory function of maternally transmitted Plasmodium falciparum-specific antibodies in Gabonese newborns.

An analysis of Plasmodium falciparum-specific antibodies was performed in pairs of maternal and cord sera from Gabon, a region endemic for malaria. All paired sera (n = 59) had P. falciparum-specific antibodies. Immunofluorescence assays detected parasite-specific IgG1, IgG2, and IgG3 in 100% of the tested pairs (n = 26) and IgG4 in 42% of them. The titers of specific IgG2 and IgG3 were significantly lower in cord than in maternal sera. All maternal sera had specific IgM. Of the seven P. falciparum-IgM positive cord sera, six were associated with malaria-related histological placental changes (MRHPC). In addition, higher titers of specific IgG1 in maternal and cord sera and of specific IgG3 in cord sera were associated with MRHPC. Similar P. falciparum antigens were recognized by cord and corresponding maternal sera in radioimmunoprecipitation and Western blot assays (n = 40). Sixteen of 20 cord sera and 15 of 20 paired maternal sera significantly inhibited in vitro parasite growth. The extent of inhibition did not correlate with the titer of specific antibodies. These data confirm the very effective placental transfer of anti-malarial antibodies. The presence of IgM in some cord sera raise the question of intrauterine sensitization to malaria antigens.

Optimization of natural killer T cell-mediated immunotherapy in cancer using cell-based and nanovector vaccines.

α-Galactosylceramide (α-GalCer) represents a new class of immune stimulators and vaccine adjuvants that activate type I natural killer T (NKT) cells to swiftly release cytokines and to exert helper functions for acquired immune responses. This unique property prompted clinicians to exploit the antitumor potential of NKT cells. Here, we review the effects of α-GalCer in (pre)clinics and discuss current and future strategies that aim to optimize NKT cell-mediated antitumor therapy, with a particular focus on cell-based and nanovector vaccines.

Oxidative stress-mediated iNKT-cell activation is involved in COPD pathogenesis.

Chronic obstructive pulmonary disease (COPD) is a major clinical challenge mostly due to cigarette smoke (CS) exposure. Invariant natural killer T (iNKT) cells are potent immunoregulatory cells that have a crucial role in inflammation. In the current study, we investigate the role of iNKT cells in COPD pathogenesis. The frequency of activated NKT cells was found to be increased in peripheral blood of COPD patients relative to controls. In mice chronically exposed to CS, activated iNKT cells accumulated in the lungs and strongly contributed to the pathogenesis. The detrimental role of iNKT cells was confirmed in an acute model of oxidative stress, an effect that depended on interleukin (IL)-17. CS extracts directly activated mouse and human dendritic cells (DC) and airway epithelial cells (AECs) to trigger interferonγ and/or IL-17 production by iNKT cells, an effect ablated by the anti-oxidant N-acetylcystein. In mice, this treatment abrogates iNKT-cell accumulation in the lung and abolished the development of COPD. Together, activation of iNKT cells by oxidative stress in DC and AECs participates in the development of experimental COPD, a finding that might be exploited at a therapeutic level.