Investigation of the role of SLC11A1 gene in goats immune response to infenction by the Mycobacterium Avium subspecies Paratuberculosis

The SLC11A1 protein (solute carrier family 11 member A1) is located in phagolysosomal membrane during macrophages activation. The activity of this protein has been associated with the immune response to infection by intracellular parasites. More specifically, studies link the presence of polymorphic microsatellite regions consisting of different numbers of guanine/thymine repeats [(GT)n] in the 3΄ untranslated region (3΄ UTR) of the gene, with resistance or susceptibility to intracellular infections in various species. The purpose of this study was to investigate the role of these polymorphisms in shaping the immune response of goats to Mycobacterium avium subspecies paratuberculosis (MAP) infection. Initially, we studied the expression of the SLC11A1 gene at mRNA and protein level following infection by MAP and investigated whether this change is associated with one of the 3΄ UTR polymorphisms of the specific gene. Then we assessed the immune response of macrophages following infection with MAP, through studying the expression of mRNA of one pro-inflammatory cytokine interleukin 1α (IL-1α) and one anti-inflammatory cytokine, interleukin 10 (IL-10). Furthermore, we investigated the functional role of polymorphic regions of 3΄ UTR of SLC11A1 gene using constructed plasmids containing the two polymorphic regions and expressing the luciferase reporter gene. Transient transfections were made in murine macrophage cell line using for stimulation MAP, PPD, IFN-gamma, LPS separately and in combination. In silico analysis was also performed to predict motifs that their action may be affected in post-transcriptional level by polymorphisms in the 3΄ UTR of the SLC11A1 gene. The results regarding the nucleotide sequence of the 3΄ UTR of the SLC11A1 gene are in agreement with previous findings in terms of their structure and the two polymorphic microsatellite regions A and B. Strong association was recorded for the genotype B7/B7 with elevated level of mRNA of SLC11A1 gene at 3 hours after stimulation with MAP, compared to goat monocytes with the other two genotypes bearing B8 allele. These increased expression levels of the gene appears to be followed by increased expression of SLC11A1 protein 3 and 24 hours after stimulation with MAP, compared to the heterozygous genotype. The difference of expression between genotypes B7/7 and B7/8, B8/8 continues further by increased mRNA concentration of IL-1a gene, 24 hours after stimulation with MAP. In contrast to the results of the first part of this study, plasmid B (GT16-GT8) was found to generate the strongest enzyme activity. Furthermore the biggest difference in luciferase activity after stimulation (non-stimulated, LPS, IFN-γ - PPD, IFN-γ - LPS - PPD) was recorded for plasmids B (GT16-GT8) compared to C (GT15-GT7). The genotypes of these two plasmids have respectively the highest and smallest (24 and 22) total number of dinoucleotides amongst the plasmids constructed here. The above experiments show that the A area alone, especially containing GT16 polymorphisms, and in combination with specific polymorphisms GT8 in the B area, may be a potent transcriptional enhancer. In the last part of this study we predicted five sites of transcriptional regulatory elements and we detected 85 miRNAs that can potentially bind and modulate the stability of transcripts of this gene. It can be concluded that the results of this study demonstrated that polymorphisms of 3΄UTR of the SLC11A1 gene are functional. The latter was confirmed at the transcriptional, post-transcriptional and translational level. Indeed their effect individually indicates strong stimulation of GT16 polymorphism of area A combined with a particular polymorphism of GT8 at B area. In transcriptional and translational level the effect of such specific polymorphisms in reference standard used, appears to differ highlighting very strong stimulating effect on the polymorphism GT7 of area B in homozygous condition (B7/7).Η πρωτεΐνη SLC11A1 (solute carrier family 11 member A1) τοποθετείται στη φαγολυσοσωμική μεμβράνη κατά την ενεργοποίηση των μακροφάγων. Η δράση της έχει συνδεθεί με την ανοσοποιητική απόκριση του οργανισμού σε μολύνσεις από ενδοκυττάρια παράσιτα. Πιο συγκεκριμένα, μελέτες συνδέουν την παρουσία μικροδορυφορικών πολυμορφικών περιοχών που συνιστούνται από διαφόρου αριθμού επαναλήψεις γουανίνης/θυμίνης στην 3΄ μη μεταφραζόμενη περιοχή του γονιδίου, με ανθεκτικότητα ή ευαισθησία σε ενδοκυττάριες λοιμώξεις σε διάφορα ζωικά είδη. Σκοπός της παρούσας μελέτης είναι να διερευνηθεί ο ρόλος αυτών των πολυμορφισμών στη διαμόρφωση της ανοσοποιητικής απόκρισης των αιγών στη λοίμωξη από το βακτήριο Mycobacterium avium υποείδος paratuberculosis (MAP). Αρχικά μελετήθηκε η έκφραση του γονιδίου SLC11A1 σε επίπεδο mRNA και πρωτεΐνης ύστερα από μόλυνση με MAP και κατά πόσο αυτή συνδέεται με κάποιον από τους πολυμορφισμούς της 3΄ UTR του συγκεκριμένου γονιδίου. Έπειτα έγινε προσπάθεια διερεύνησης της ανοσοποιητικής απόκρισης των μακροφάγων ύστερα από μόλυνση με MAP, μέσω της μελέτης έκφρασης του γονιδίου μίας προφλεγμονώδους και μίας αντιφλεγμωνώδους κυτταροκίνης, της ιντερλευκίνης 1α (IL-1α) και της ιντερλευκίνης 10 (IL-10) αντίστοιχα. Επιπλέον, έγινε διερεύνηση της λειτουργικής επίδρασης των πολυμορφικών περιοχών της 3΄ UTR στην έκφραση του γονιδίου SLC11A1 με την κατασκευή πλασμιδίων που έφεραν τις δύο πολυμορφικές περιοχές σε συνδυασμό και εκφράζουν ως γονίδο αναφοράς αυτό της λουσιφεράσης. Έγιναν παροδικές διαμολύνσεις κυτταρικής σειράς μακροφάγων ποντικού και διεγέρθηκαν με MAP, PPD, IFN-γ και LPS, καθώς και με συνδυασμό τους, και προσδιορίστηκε η ενεργότητα του γονιδίου της λουσιφεράσης. Ακολούθησε στατιστική ανάλυση και in silico μελέτη πρόβλεψης προτύπων των οποίων η δράση επηρεάζεται σε μεταμεταγραφικό επίπεδο από τον πολυμορφισμό στην 3΄ UTR του γονιδίου SLC11A1. Το αποτελέσμα ως προς τον προσδιορισμό της νουκλεοτιδικής αλληλουχίας της 3΄ UTR του γονιδόυ SLC11A1 συμφωνεί με προηγούμενα ευρύματα αναφορικά με τη δομή όσο και και τη σύσταση των δύο μικροδορυφορικών πολυμορφικών περιοχών Α και Β. Καταγράφεται ισχυρή συσχέτιση του γονοτύπου B7/B7 με αυξημένο επίπεδο mRNA του γονιδίου SLC11A1 3 ώρες μετά τη διέγερση με MAP, σε μονοκύτταρα αιγών σε σύγκριση με τους δύο άλλους γονότυπους που φέρουν το αλλήλιο Β8. Η αύξηση της συγκέντρωσης του mRNA φαίνεται ότι ακολουθείται από αυξημένη έκφραση της πρωτεΐνης SLC11A1 3 και 24 ώρες μετά τη διέγερση με MAP, σε σχέση με τον ετερόζυγο γονότυπο. Αυτή η διαφοράς της έκφρασης μεταξύ γονοτύπων Β7/7 και Β7/8, Β8/8 ισχύει σε επίπεδο mRNA για το γονίδιο IL-1α, 24 ώρες μετά τη διεγερση με MAP. Στο δεύτερο μέρος αυτής της μελέτης διερευνήθηκε, αν η 3'UTR του γονιδίου SLC11A1 της αίγας μπορεί να δράσει ως λειτουργικός ενισχυτής του γονιδίου και αν η λειτουργία αυτή αλλάζει αναλόγως με τον αριθμό των επαναλήψεων GT. Αναφορικά με αυτό το θέμα προέκυψε σε αντίθεση με τα αποτελέσματα του πρώτου μέρους, ότι το πλασμίδιο Β (GT16-GT8) που εκδήλωσε την ισχυρότερη ενζυμική ενεργότητα παρουσιάζει τη μεγαλύτερη διακυμάνση ύστερα από τη διέγερση. Επίσης, μπορούμε να σημειώσουμε ότι τις μεγαλύτερες διαφορές με τους περισσότερους διεγέρτες (αδιέγερτα, LPS, IFN-γ – PPD, IFN-γ – LPS – PPD) φαίνεται να τις έχουν τα πλασμίδια Β (GT16-GT8) με C (GT15-GT7), τα οποία έχουν και τους ακραίους γονότυπους με συνολικό αριθμό επαναλήψεων και των δύο περιοχών, 24 και 22 δινουκλεοτιδίων αντίστοιχα. Από τα παραπάνω πειράματα προκύπτει ότι η περιοχή Α από μόνη της ιδίως με τον πολυμορφισμό GT16, αλλά και σε συνδυασμό ειδικά με τον πολυμορφισμό GT8 της περιοχής Β, αποτελεί ισχυρό ενισχυτή μεταγραφής. Από την in silico μελέτη πρόβλεψης στοιχείων βρέθηκαν πέντε σημεία πρόσδεσης ρυθμιστικών μεταγραφικών στοιχείων και 85 miRNAs τα οποία μπορούν δυνητικά να προσδεθούν και να ρυθμίσουν τη σταθερότητα των μεταγράφων του γονιδίου αυτού. Συμπερασματικά μπορεί να αναφερθεί ότι από το αποτέλεσμα της παρούσας μελέτης αποδεικνύεται ότι οι πολυμορφισμοί της 3΄UTR του γονιδίου SLC11A1 είναι λειτουργικοί. Το τελευταίο επαληθεύτηκε σε μεταγραφικό, μέτα-μεταγραφικό και μεταφραστικό επίπεδο. Μάλιστα η επίδρασή τους μεμονωμένα υποδηλώνει έντονη διέγερση από τον πολυμορφισμό GT16 της περιοχής Α ιδίως σε συνδυασμό με τον πολυμορφισμό GT8 της Β. Σε μεταγραφικό και μεταφραστικό επίπεδο η επίδραση που έχουν οι συγκεκριμένοι πολυμορφισμοί στο πρότυπο αναφοράς που χρησιμοποιήθηκε, φαίνεται να διαφοροποιούνται αναδεικνύοντας πολύ ισχυρή διεγερτική επίδραση για τον πολυμορφισμό GT7 της περιοχής Β σε ομόζυγη κατάσταση (πολυμορφισμός Β7/7)

Transcription factor ATF-3 regulates allele variation phenotypes of the human SLC11A1 gene

Genetic polymorphisms in the human solute carrier family 11 member 1 (SLC11A1) gene predispose to susceptibility to infectious/inflammatory diseases and cancer. Human susceptibility to these diseases exhibits allelic association with a polymorphic regulatory Z-DNA-forming microsatellite of a (GT/AC)n repeat. The carriage of different alleles may influence chromatin remodeling and accessibility by transcription factors. Of particular importance is the binding site for the Activating Protein 1 (AP-1) elements, (ATF-3 and c-Jun), adjacent to the 5’ sequence of the Z-DNA-forming polymorphism. The aim of the study was to characterize the transcriptional mechanisms controlling different alleles of SLC11A1 expression by ATF-3 and c-Jun. Allele 2, [T(GT)(5)AC(GT)(5)AC(GT)(10)GGCAGA(G)(6)], and Allele 3, [T(GT)(5)AC(GT)(5)AC(GT)(9)GGCAGA(G)(6)], were subcloned into the PGL2Basic vector. Transient transfections of THP-1 cells with the constructs, in the presence or absence of pATF-3 were preformed. Luciferase expression was determined. To document the recruitment of ATF-3 and c-Jun, to the polymorphic promoter alleles in vivo, we performed ChIP assays with transient transfected THP-1 cells treated with or without lipopolyssacharides. Our data documented that ATF-3 suppresses the transcriptional activation of Allele-3, and this suppression is enhanced in the presence of lipopolyssacharides. Our findings suggest that ATF-3 and c-Jun may influence heritable variation in SLC11A1-dependent innate resistance to infection and inflammation both within and between populations

Alteration of L-Dopa decarboxylase expression in SARS-CoV-2 infection and its association with the interferon-inducible ACE2 isoform

International audienceL-Dopa decarboxylase ( DDC ) is the most significantly co-expressed gene with ACE2 , which encodes for the SARS-CoV-2 receptor a ngiotensin- c onverting e nzyme 2 and the interferon-inducible truncated isoform dACE2 . Our group previously showed the importance of DDC in viral infections. We hereby aimed to investigate DDC expression in COVID-19 patients and cultured SARS-CoV-2-infected cells, also in association with ACE2 and dACE2 . We concurrently evaluated the expression of the viral infection- and interferon-stimulated gene ISG56 and the immune-modulatory, hypoxia-regulated gene EPO . Viral load and mRNA levels of DDC , ACE2 , dACE2 , ISG56 and EPO were quantified by RT-qPCR in nasopharyngeal swab samples from COVID-19 patients, showing no or mild symptoms, and from non-infected individuals. Samples from influenza-infected patients were analyzed in comparison. SARS-CoV-2-mediated effects in host gene expression were validated in cultured virus-permissive epithelial cells. We found substantially higher gene expression of DDC in COVID-19 patients (7.6-fold; p = 1.2e-13) but not in influenza-infected ones, compared to non-infected subjects. dACE2 was more elevated (2.9-fold; p = 1.02e-16) than ACE2 (1.7-fold; p = 0.0005) in SARS-CoV-2-infected individuals. ISG56 (2.5-fold; p = 3.01e-6) and EPO (2.6-fold; p = 2.1e-13) were also increased. Detected differences were not attributed to enrichment of specific cell populations in nasopharyngeal tissue. While SARS-CoV-2 virus load was positively associated with ACE2 expression (r≥0.8, p<0.001), it negatively correlated with DDC , dACE2 (r≤−0.7, p<0.001) and EPO (r≤−0.5, p<0.05). Moreover, a statistically significant correlation between DDC and dACE2 expression was observed in nasopharyngeal swab and whole blood samples of both COVID-19 and non-infected individuals (r≥0.7). In VeroE6 cells, SARS-CoV-2 negatively affected DDC , ACE2 , dACE2 and EPO mRNA levels, and induced cell death, while ISG56 was enhanced at early hours post-infection. Thus, the regulation of DDC , dACE2 and EPO expression in the SARS-CoV-2-infected nasopharyngeal tissue is possibly related with an orchestrated antiviral response of the infected host as the virus suppresses these genes to favor its propagation

Alteration of L-Dopa decarboxylase expression in SARS-CoV-2 infection and its association with the interferon-inducible ACE2 isoform

L-Dopa decarboxylase (DDC) is the most significantly co-expressed gene with ACE2, which encodes for the SARS-CoV-2 receptor angiotensin-converting enzyme 2 and the interferon-inducible truncated isoform dACE2. Our group previously showed the importance of DDC in viral infections. We hereby aimed to investigate DDC expression in COVID-19 patients and cultured SARS-CoV-2-infected cells, also in association with ACE2 and dACE2. We concurrently evaluated the expression of the viral infection- and interferon-stimulated gene ISG56 and the immune-modulatory, hypoxia-regulated gene EPO. Viral load and mRNA levels of DDC, ACE2, dACE2, ISG56 and EPO were quantified by RT-qPCR in nasopharyngeal swab samples from COVID-19 patients, showing no or mild symptoms, and from non-infected individuals. Samples from influenza-infected patients were analyzed in comparison. SARS-CoV-2-mediated effects in host gene expression were validated in cultured virus-permissive epithelial cells. We found substantially higher gene expression of DDC in COVID-19 patients (7.6-fold; p = 1.2e-13) but not in influenza-infected ones, compared to non-infected subjects. dACE2 was more elevated (2.9-fold; p = 1.02e-16) than ACE2 (1.7-fold; p = 0.0005) in SARS-CoV-2-infected individuals. ISG56 (2.5-fold; p = 3.01e-6) and EPO (2.6-fold; p = 2.1e-13) were also increased. Detected differences were not attributed to enrichment of specific cell populations in nasopharyngeal tissue. While SARS-CoV-2 virus load was positively associated with ACE2 expression (r &gt;= 0.8, p&lt;0.001), it negatively correlated with DDC, dACE2 (r &lt;=-0.7, p&lt;0.001) and EPO (r &lt;=-0.5, p&lt;0.05). Moreover, a statistically significant correlation between DDC and dACE2 expression was observed in nasopharyngeal swab and whole blood samples of both COVID-19 and non-infected individuals (r &gt;= 0.7). In VeroE6 cells, SARS-CoV-2 negatively affected DDC, ACE2, dACE2 and EPO mRNA levels, and induced cell death, while ISG56 was enhanced at early hours post-infection. Thus, the regulation of DDC, dACE2 and EPO expression in the SARS-CoV-2-infected nasopharyngeal tissue is possibly related with an orchestrated antiviral response of the infected host as the virus suppresses these genes to favor its propagation

Development and characterization of DNAzyme candidates demonstrating significant efficiency against human rhinoviruses

BACKGROUND: Infections with human rhinoviruses (RVs) are responsible for millions of common cold episodes and the majority of asthma exacerbations, especially in childhood. No drugs specifically targeting RVs are available. OBJECTIVE: We sought to identify specific anti-RV molecules based on DNAzyme technology as candidates to a clinical study. METHODS: A total of 226 candidate DNAzymes were designed against 2 regions of RV RNA genome identified to be sufficiently highly conserved between virus strains (ie, the 5'-untranslated region and cis-acting replication element) by using 3 test strains: RVA1, RVA16, and RVA29. All DNAzymes were screened for their cleavage efficiency against in vitro-expressed viral RNA. Those showing any catalytic activity were subjected to bioinformatic analysis of their reverse complementarity to 322 published RV genomic sequences. Further molecular optimization was conducted for the most promising candidates. Cytotoxic and off-target effects were excluded in HEK293 cell-based systems. Antiviral efficiency was analyzed in infected human bronchial BEAS-2B cells and ex vivo-cultured human sinonasal tissue. RESULTS: Screening phase-generated DNAzymes characterized by either good catalytic activity or by high RV strain coverage but no single molecule represented a satisfactory combination of those 2 features. Modifications in length of the binding domains of 2 lead candidates, Dua-01(-L12R9) and Dua-02(-L10R11), improved their cleavage efficiency to an excellent level, with no loss in eminent strain coverage (about 98%). Both DNAzymes showed highly favorable cytotoxic/off-target profiles. Subsequent testing of Dua-01-L12R9 in BEAS-2B cells and sinonasal tissue demonstrated its significant antiviral efficiency. CONCLUSIONS: Effective and specific management of RV infections with Dua-01-L12R9 might be useful in preventing asthma exacerbations, which should be verified by clinical trials

Extensively Hydrolyzed Hypoallergenic Infant Formula with Retained T Cell Reactivity

Background: Immunoglobulin E (IgE)-mediated cow’s milk allergy (CMA) can be life-threatening and affects up to 3% of children. Hypoallergenic infant formulas based on hydrolyzed cow’s milk protein are increasingly considered for therapy and prevention of cow’s milk allergy. The aim of this study was to investigate the allergenic activity and ability to induce T cell and cytokine responses of an infant formula based on extensively hydrolyzed cow’s milk protein (whey) (eHF, extensively hydrolyzed formula) supplemented with Galactooligosaccharides (GOS) and Limosilactobacillus fermentum CECT5716 (LF) to determine its suitability for treatment and prevention of CMA. Methods: eHF and standard protein formula based on intact cow’s milk proteins (iPF) with or without Galactooligosaccharide (GOS) and Limosilactobacillus fermentum CECT5716 (LF) were investigated with allergen-specific antibodies and tested for IgE reactivity and allergenic activity in basophil degranulation assays with sera from cow’s milk (CM)-allergic infants/children. Their ability to stimulate T cell proliferation and cytokine secretion in cultured peripheral blood mononuclear cells (PBMC) from CM-allergic infants and children was studied with a FACS-based carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution assay and xMAP Luminex fluorescent bead-based technology, respectively. Results: An eHF supplemented with GOS and LF exhibiting almost no IgE reactivity and allergenic activity was identified. This eHF induced significantly lower inflammatory cytokine secretion as compared to an intact protein-based infant formula but retained T cell reactivity. Conclusions: Due to strongly reduced allergenic activity and induction of inflammatory cytokine secretion but retained T cell reactivity, the identified eHF may be used for treatment and prevention of CMA by induction of specific T cell tolerance