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Eukaryote plasma membranes protect cells from chemical attack. Xenobiotics, taken up through passive diffusion, accumulate in the membranes, where they are captured by transporters, among which P-glycoproteins (Pgps). In nematodes such as Haemonchus contortus, eggshells and cuticles provide additional protective barriers against xenobiotics. Little is known about the role of these structures in the transport of chemical molecules. Pgps, members of the ABC transporter family, are present in eggshells and cuticles. Changes in the activity of these proteins have also been correlated with alterations in lipids, such as cholesterol content, in eggshells. However, the cellular mechanisms underlying these effects remain unclear. We show here that an experimental decrease in the cholesterol content of eggshells of Haemonchus contortus, with Methyl-beta-CycloDextrin (MβCD), results in an increase in membrane fluidity, favouring Pgp activity and leading to an increase in resistance to anthelmintics. This effect is modulated by the initial degree of anthelminthic resistance of the eggs. These results suggest that eggshell fluidity plays a major role in the modulation of Pgp activity. They confirm that Pgp activity is highly influenced by the local microenvironment, in particular sterols, as observed in some vertebrate models. Thus, eggshell barriers could play an active role in the transport of xenobiotics.

Abstract Background Telomerase activation, a critical step in cell immortalization and oncogenesis, is partly regulated by alternative splicing. In this study, we aimed to use the Marek's disease virus (MDV) T-cell lymphoma model to evaluate TERT regulation by splicing during lymphomagenesis in vivo, from the start point to tumor establishment. Results We first screened cDNA libraries from the chicken MDV lymphoma-derived MSB-1 T- cell line, which we compared with B (DT40) and hepatocyte (LMH) cell lines. The chTERT splicing pattern was cell line-specific, despite similar high levels of telomerase activity. We identified 27 alternative transcripts of chicken TERT (chTERT). Five were in-frame alternative transcripts without in vitro telomerase activity in the presence of viral or chicken telomerase RNA (vTR or chTR), unlike the full-length transcript. Nineteen of the 22 transcripts with a premature termination codon (PTC) harbored a PTC more than 50 nucleotides upstream from the 3' splice junction, and were therefore predicted targets for nonsense-mediated decay (NMD). The major PTC-containing alternatively spliced form identified in MSB1 (ie10) was targeted to the NMD pathway, as demonstrated by UPF1 silencing. We then studied three splicing events separately, and the balance between in-frame alternative splice variants (d5f and d10f) plus the NMD target i10ec and constitutively spliced chTERT transcripts during lymphomagenesis induced by MDV indicated that basal telomerase activity in normal T cells was associated with a high proportion of in-frame non functional isoforms and a low proportion of constitutively spliced chTERT. Telomerase upregulation depended on an increase in active constitutively spliced chTERT levels and coincided with a switch in alternative splicing from an in-frame variant to NMD-targeted variants. Conclusions TERT regulation by splicing plays a key role in telomerase upregulation during lymphomagenesis, through the sophisticated control of constitutive and alternative splicing. Using the MDV T-cell lymphoma model, we identified a chTERT splice variant as a new NMD target.

Salmonella enterica subspecies enterica serovar Typhimurium is an intracellular pathogen that invades and colonizes the intestinal epithelium. Following bacterial invasion, Salmonella is enclosed within a membrane-bound vacuole known as a Salmonella -containing vacuole (SCV). However, a subset of Salmonella has the capability to prematurely rupture the SCV and escape, resulting in Salmonella hyper-replication within the cytosol of epithelial cells. A recently published RNA-seq study provides an overview of cytosolic and vacuolar upregulated genes and highlights pagN vacuolar upregulation. Here, using transcription kinetics, protein production profile, and immunofluorescence microscopy, we showed that PagN is exclusively produced by Salmonella in SCV. Gentamicin protection and chloroquine resistance assays were performed to demonstrate that deletion of pagN affects Salmonella replication by affecting the cytosolic bacterial population. This study presents the first example of a Salmonella virulence factor expressed within the endocytic compartment, which has a significant impact on the dynamics of Salmonella cytosolic hyper-replication.

Neutrophils can be beneficial or deleterious during tuberculosis (TB). Based on the expression of MHC-II and programmed death ligand 1 (PD-L1), we distinguished two functionally and transcriptionally distinct neutrophil subsets in the lungs of mice infected with mycobacteria. Inflammatory [MHC-II - , PD-L1 lo ] neutrophils produced inflammasome-dependent IL-1β in the lungs in response to virulent mycobacteria and "accelerated" deleterious inflammation, which was highly exacerbated in IFN-γR -/- mice. Regulatory [MHC-II + , PD-L1 hi ] neutrophils "brake" inflammation by suppressing T-cell proliferation and IFN-γ production. Such beneficial regulation, which depends on PD-L1, is controlled by IFN-γR signaling in neutrophils. The hypervirulent HN878 strain from the Beijing genotype curbed PD-L1 expression by regulatory neutrophils, abolishing the braking function and driving deleterious hyperinflammation in the lungs. These findings add a layer of complexity to the roles played by neutrophils in TB and may explain the reactivation of this disease observed in cancer patients treated with anti-PD-L1., (© 2024 Doz-Deblauwe et al.)

Ankyrin proteins (ANKRD) are key mediators linking membrane and sub-membranous cytoskeletal proteins. Recent findings have highlighted a new role of ANKRD31 during spermatogenesis, elucidating its involvement in meiotic recombination and male germ cell progression. Following testicular differentiation, spermatozoa (SPZ) enter into the epididymis, where they undergo several biochemical and enzymatic changes. The epididymal epithelium is characterized by cell-to-cell junctions that are able to form the blood-epididymal barrier (BEB). This intricate epithelial structure provides the optimal microenvironment needed for epididymal sperm maturation. To date, no notions have been reported regarding a putative role of ANKRD31 in correct BEB formation. In our work, we generated an Ankrd31 knockout male mouse model ( Ankrd31 -/- ) and characterized its reproductive phenotype. Ankrd31 -/- mice were infertile and exhibited oligo-astheno-teratozoospermia (a low number of immotile SPZ with abnormal morphological features). In addition, a complete deregulation of BEB was found in Ankrd31 -/- , due to cell-to-cell junction anomalies. In order to suggest that BEB deregulation may depend on Ankrd31 gene deletion, we showed the physical interaction among ANKRD31 and some epithelial junction proteins in wild-type (WT) epididymides. In conclusion, the current work shows a key role of ANKRD31 in the control of germ cell progression as well as sperm and epididymal integrity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Manfrevola, Martinez, Coutton, Rocco, Reynaud, Le Vern, Froment, Beauclair, Aubert, Pierantoni, Chianese and Guillou.)

Neutrophils that reside in the bone marrow are swiftly recruited from circulating blood to fight infections. For a long time, these first line defenders were considered as microbe killers. However their role is far more complex as cross talk with T cells or dendritic cells have been described for human or mouse neutrophils. In cattle, these new roles are not documented yet. We identified a new subset of regulatory neutrophils that is present in the mouse bone marrow or circulate in cattle blood under steady state conditions. These regulatory neutrophils that display MHC-II on the surface are morphologically indistinguishable from classical MHC-II neg neutrophils. However MHC-II pos and MHC-II neg neutrophils display distinct transcriptomic profiles. While MHC-II neg and MHC-II pos neutrophils display similar bacterial phagocytosis or killing activity, MHC-II pos only are able to suppress T cell proliferation under contact-dependent mechanisms. Regulatory neutrophils are highly enriched in lymphoid organs as compared to their MHC-II neg counterparts and in the mouse they express PDL-1, an immune checkpoint involved in T-cell blockade. Our results emphasize neutrophils as true partners of the adaptive immune response, including in domestic species. They open the way for discovery of new biomarkers and therapeutic interventions to better control cattle diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Rambault, Doz-Deblauwe, Le Vern, Carreras, Cunha, Germon, Rainard, Winter and Remot.)

Flow cytometry and magnetic bead technology enable the separation of cell populations with the highest degree of purity. Here, we describe protocols to sort bovine neutrophils from blood, the labeling and sorting, including gating strategies. We also provide advice to preserve neutrophil viability and detail a protocol to measure phagocytosis and oxidative species production.

Salmonella Typhimurium expresses on its outer membrane the protein Rck which interacts with the epidermal growth factor receptor (EGFR) of the plasma membrane of the targeted host cells. This interaction activates signaling pathways, leading to the internalization of Salmonella . Since EGFR plays a key role in cell proliferation, we sought to determine the influence of Rck mediated infection on the host cell cycle. By analyzing the DNA content of uninfected and infected cells using flow cytometry, we showed that the Rck-mediated infection induced a delay in the S-phase (DNA replication phase) of the host cell cycle, independently of bacterial internalization. We also established that this Rck-dependent delay in cell cycle progression was accompanied by an increased level of host DNA double strand breaks and activation of the DNA damage response. Finally, we demonstrated that the S-phase environment facilitated Rck-mediated bacterial internalization. Consequently, our results suggest that Rck can be considered as a cyclomodulin with a genotoxic activity., (Copyright © 2020 Mambu, Barilleau, Fragnet-Trapp, Le Vern, Olivier, Sadrin, Grépinet, Taieb, Velge and Wiedemann.)

Cryptosporidium parvum causes diarrhea in infants under 5 years, in immunosuppressed individuals or in young ruminants. This parasite infects the apical side of ileal epithelial cells where it develops itself and induces inflammation. Antimicrobial peptides (AMPs) are part of the innate immune response, playing a major role in the control of the acute phase of C. parvum infection in neonates. Intestinal AMP production in neonates is characterized by high expressions of Cathelicidin Related Antimicrobial Peptide (CRAMP), the unique cathelicidin in mice known to fight bacterial infections. In this study, we investigated the role of CRAMP during cryptosporidiosis in neonates. We demonstrated that sporozoites are sensitive to CRAMP antimicrobial activity. However, during C. parvum infection the intestinal expression of CRAMP was significantly and selectively reduced, while other AMPs were upregulated. Moreover, despite high CRAMP expression in the intestine of neonates at homeostasis, the depletion of CRAMP did not worsen C. parvum infection. This result might be explained by the rapid downregulation of CRAMP induced by infection. However, the exogenous administration of CRAMP dampened the parasite burden in neonates. Taken together these results suggest that C. parvum impairs the production of CRAMP to subvert the host response, and highlight exogenous cathelicidin supplements as a potential treatment strategy.

In this study, we aimed to determine the origin of the difference, in terms of anti-Müllerian hormone production, existing between the bovine and porcine ovaries. We first confirmed by quantitative real-time-Polymerase-Chain Reaction, ELISA assay and immunohistochemistry that anti-Müllerian hormone mRNA and protein production are very low in porcine ovarian growing follicles compared to bovine ones. We then have transfected porcine and bovine granulosa cells with vectors containing the luciferase gene driven by the porcine or the bovine anti-Müllerian hormone promoter. These transfection experiments showed that the porcine anti-Müllerian hormone promoter is less active and less responsive to bone morphogenetic protein stimulations than the bovine promoter in both porcine and bovine cells. Moreover, bovine but not porcine granulosa cells were responsive to bone morphogenetic protein stimulation after transfection of a plasmidic construction including a strong response element to the bone morphogenetic proteins (12 repetitions of the GCCG sequence) upstream of the luciferase reporter gene. We also showed that SMAD6, an inhibitor of the SMAD1-5-8 pathway, is strongly expressed in porcine compared to the bovine granulosa cells. Overall, these results suggest that the low expression of anti-Müllerian hormone in porcine growing follicles is due to both a lack of activity/sensitivity of the porcine anti-Müllerian hormone promoter, and to the lack of responsiveness of porcine granulosa cells to bone morphogenetic protein signaling, potentially due to an overexpression of SMAD6 compared to bovine granulosa cells. We propose that the low levels of anti-Müllerian hormone in the pig would explain the poly-ovulatory phenotype in this species., (© The Author(s) 2020. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Coccidia are obligate intracellular protozoan parasites responsible for human and veterinary diseases. Eimeria tenella, the aetiologic agent of caecal coccidiosis, is a major pathogen of chickens. In Toxoplasma gondii, some kinases from the rhoptry compartment (ROP) are key virulence factors. ROP kinases hijack and modulate many cellular functions and pathways, allowing T. gondii survival and development. E. tenella's kinome comprises 28 putative members of the ROP kinase family; most of them are predicted, as pseudokinases and their functions have never been characterised. One of the predicted kinase, EtROP1, was identified in the rhoptry proteome of E. tenella sporozoites. Here, we demonstrated that EtROP1 is active, and the N-terminal extension is necessary for its catalytic kinase activity. Ectopic expression of EtROP1 followed by co-immunoprecipitation identified cellular p53 as EtROP1 partner. Further characterisation confirmed the interaction and the phosphorylation of p53 by EtROP1. E. tenella infection or overexpression of EtROP1 resulted both in inhibition of host cell apoptosis and G0/G1 cell cycle arrest. This work functionally described the first ROP kinase from E. tenella and its noncanonical structure. Our study provides the first mechanistic insight into host cell apoptosis inhibition by E. tenella. EtROP1 appears as a new candidate for coccidiosis control., (© 2019 The Authors Cellular Microbiology Published by John Wiley & Sons Ltd.)

Marek's disease virus (MDV) is a highly contagious alphaherpesvirus that infects chickens and causes a deadly neoplastic disease. We previously demonstrated that MDV infection arrests cells in S phase and that the tegument protein VP22 plays a major role in this process. In addition, expression of VP22 induces double-strand breaks (DSBs) in the cellular DNA, suggesting that DNA damage and the associated cellular response might be favorable for the MDV life cycle. Here, we addressed the role of DNA damage in MDV replication and pathogenesis. We demonstrated that MDV induces DSBs during lytic infection in vitro and in the peripheral blood mononuclear cells of infected animals. Intriguingly, we did not observe DNA damage in latently infected MDV-induced lymphoblastoid cells, while MDV reactivation resulted in the onset of DNA lesions, suggesting that DNA damage and/or the resulting DNA damage response might be required for efficient MDV replication and reactivation. In addition, reactivation was significantly enhanced by the induction of DNA damage using a number of chemicals. Finally, we used recombinant viruses to show that VP22 is required for the induction of DNA damage in vivo and that this likely contributes to viral oncogenesis. IMPORTANCE Marek's disease virus is an oncogenic alphaherpesvirus that causes fatal T-cell lymphomas in chickens. MDV causes substantial losses in the poultry industry and is also used in small-animal models for virus-induced tumor formation. DNA damage not only is implicated in tumor development but also aids in the life cycle of several viruses; however, its role in MDV replication, latency, and reactivation remains elusive. Here, we demonstrate that MDV induces DNA lesions during lytic replication in vitro and in vivo DNA damage was not observed in latently infected cells; however, it was reinitiated during reactivation. Reactivation was significantly enhanced by the induction of DNA damage. Recombinant viruses that lacked the ability to induce DNA damage were defective in their ability to induce tumors, suggesting that DNA damage might also contribute to cellular transformation processes leading to MDV lymphomagenesis., (Copyright © 2017 American Society for Microbiology.)

The Salmonella Rck outer membrane protein binds to the cell surface, which leads to bacterial internalization via a Zipper mechanism. This invasion process requires induction of cellular signals, including phosphorylation of tyrosine proteins, and activation of c-Src and PI3K, which arises as a result of an interaction with a host cell surface receptor. In this study, epidermal growth factor receptor (EGFR) was identified as the cell signaling receptor required for Rck-mediated adhesion and internalization. First, Rck-mediated adhesion and internalization were shown to be altered when EGFR expression and activity were modulated. Then, immunoprecipitations were performed to demonstrate the Rck-EGFR interaction. Furthermore, surface plasmon resonance biosensor and homogeneous time-resolved fluorescence technologies were used to demonstrate the direct interaction of Rck with the extracellular domain of human EGFR. Finally, our study strongly suggests a noncompetitive binding of Rck and EGF to EGFR. Overall, these results demonstrate that Rck is able to bind to EGFR and thereby establish a tight adherence to provide a signaling cascade, which leads to internalization of Rck-expressing bacteria.-Wiedemann, A., Mijouin, L., Ayoub, M. A., Barilleau, E., Canepa, S., Teixeira-Gomes, A. P., Le Vern, Y., Rosselin, M., Reiter, E., Velge, P. Identification of the epidermal growth factor receptor as the receptor for Salmonella Rck-dependent invasion., (© FASEB.)

During chronic infection with Mycobacterium tuberculosis (Mtb), bacilli multiplication is constrained within lung granulomas until excessive inflammation destroys the lung. Neutrophils are recruited early and participate in granuloma formation, but excessive neutrophilia exacerbates the tuberculosis disease. Neutrophils thus appear as potential targets for therapeutic interventions, especially in patients for whom no antibiotic treatment is possible. Signals that regulate neutrophil recruitment to the lung during mycobacterial infection need to be better understood. We demonstrated here, in the mouse model, that neutrophils were recruited to the lung in two waves after intranasal infection with virulent Mtb or the live attenuated vaccine strain Bacillus Calmette Guérin (BCG). A first wave of neutrophils was swiftly recruited, followed by a subsequent adaptive wave that reached the lung together with IFN-γ- and IL-17A-producing T cells. Interestingly, the second neutrophil wave did not participate to mycobacteria control in the lung and established contacts with T cells. The adaptive wave was critically dependent on the expression of IL-17RA, the receptor for IL-17A, expressed in non-hematopoietic cells. In absence of this receptor, curtailed CXCL-1 and 5 production in the lung restrained neutrophil recruitment. CXCL-1 and 5 instillation reconstituted lung neutrophil recruitment in BCG-infected IL17RA-/- mice.

CCL20 is a chemokine with antimicrobial activity. We investigated its expression and role during neonatal cryptosporidiosis, a worldwide protozoan enteric disease leading to severe diarrhea. Surprisingly, during infection by Cryptosporidium parvum, CCL20 production by the intestine of neonatal mice is reduced by a mechanism independent both of the enteric flora and of interferon γ, a key cytokine for the resolution of this infection. However, oral administration of recombinant CCL20 to neonatal mice significantly reduced the parasite load by a mechanism that was independent of immune cell recruitment and occurred instead by direct cytolytic activity on free stages of the parasite. MiR21 functionally targets CCL20 and is upregulated during the infection, thus contributing to the downregulation of the chemokine. Our findings demonstrate for the first time the direct antiparasitic activity of CCL20 against an enteric protozoan and its downregulation during C. parvum infection, which is detrimental to parasite clearance., (© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)