Rhesus Macaque DNGR-1 And Its Alteration After Immunodeficiency Virus Infection

BackgroundDC NK lectin group receptor-1(DNGR-1),also known as C-type lectin domain family 9 member A(CLEC9A),belongs to the type II transmembrane proteins,is a member of C-type lectin receptor family.Its expression is highly restricted to a specific subset of dentritic cells(DC)that could recognize filament actin(F-actin)exposed on dead/damaged cells.DNGR-1 can mediate endocytosis and is involved in cross-presentation of exogenous antigens including dead cells and tumor associated antigens to prime cellular and humoral immunity.DNGR-1 plays an important role in the prevention and treatment of tumors and in antiviral immunity.In recent years,much attention has been focused on the structure and function of human and mouse DNGR-1,and the crucial role of DNGR-1 in specific immune responses to tumors and viral infections.Cells expressing DNGR-1 are restricted to a small subset of DCs including human BDCA3+(CD141+)DCs and mouse splenic CD8a+ DCs and peripheral CD103+ DCs and sharing similar functional capacity to that of mouse.However,human DNGR-1 is different from that of mice in the structure of ITAM-like motif in its cytoplasmic tail,the number of alternative splice variants and the form(dimer or monomer)of the molecule.Furthermore,the homology of the nucleotide and amino acid sequence between human DNGR-1 and mouse Dngr-1 is low.Rhesus macaque is genetically closely related to humans and is an important animal model for studies of vaccines and human diseases.For better use of rhesus macaques in researches on the role of DNGR-1 in human diseases and AIDS vaccines,we cloned and expressed rhesus macaque DNGR-1 gene,characterized its binding property,determined the distribution of DNGR-1mRNA and DNGR-1+ cells in the various tissues of normal rhesus macaques and examined its alteration after immunodeficiency virus infection.The findings provided a scientific basis for using rhesus macaques in human disease studies and exploration of targeted treatments of AIDS.Methods1.The nucleotide sequence of rhesus macaque DNGR-1cDNA was obtained by RT-PCR and RACE.Sequences were edited and aligned with Clustal W in Bioedit and MegAlign.MEGA 6.0 was used to construct phylogenetic trees based on nucleotide and amino acid sequences.2.C-type lectin-like domain(CTLD)of rhesus macaque DNGR-1 was amplified,cloned into the pET-32a(+)vector and expressed in the BL21(DE3)cells.The recombinant CTLD protein of rhesus macaque DNGR-1 was purified with the Ni-Agarose Resin.Female New Zealand white rabbits were immunized with recombinant CTLD protein after refolding.The blood was collected after the last boost immunization.The ORF of rhesus macaque DNGR-1 was cloned into the pcDNATM3.1D/V5-His TOPO(?)vector.HeLa cells expressing recombinant DNGR-1 were used in adherence assay.3.Apoptotic and dead cells were induced by serum deprivation,freezing-thawing or kept at room temperature for different days,membrane integrity was damaged by fixation with formaldehyde and permeabilized with saponin.Pyroptotic cells were stimulated with PMA and LPS.And then,binding of DNGR-1 to apoptotic and pyroptotic cells were examined by immunofluorescence and flow cytometry.4.Rhesus macaque DNGR-1mRNA levels were examined by nest-PCR and real-time PCR.Western blotting was used to evaluate the DNGR-1 protein levels.Localization of DNGR-1+DCs in lymphoid and non-lymphoid tissues was performed with TSA Plus Fluorescence Kits and observed under Laser Scanning Confocal Microscope.5.DNGR-1mRNA levels in lymph nodes,GALT and the blood from normal and infected rhesus macaques and humans were quantitated by real-time PCR.Furthermore,binding of DNGR-1 to apoptotic and dead cells in the blood from infected rhesus macaques and humans were examined by flow cytometry.Results1.A total of 1270bp assembled DNGR-1 nucleotide sequence including a 726bp open reading frame(ORF),a 124bp 5’-untranslated region(UTR)and a 420bp 3’-UTR was obtained.Rhesus macaque DNGR-1 was mapped to chromosome 11 and corresponded to 15739bp in the genomic DNA of Chinese rhesus macaque.The complete ORF sequence was encoded by 6 exons.The deduced DNGR-1 consists of a cytoplasmic tail,a transmembrane region,a stalk region and a CTLD.Rhesus macaque DNGR-1 gene shared 71.3%-99.3%nucleotide similarities and 54.4%-99.2%amino acid similarities with that of the other species found in GenBank(up to 2015).The similarities of nucleotide and deduced amino acid sequences to that of other primates were the highest and the lowest to rodents.Phylogenetic analysis indicated that rhesus macaque DNGR-1 gene was closely related to that of Cercopithecidae which were clustered together,and less closely to that of the members of Hominoidea which were also clustered together.However,the sequence distantly was related to that of Mus musculus and Rattus norvegicus,which formed a separate clade.2.The recombinant CTLD protein of rhesus macaque DNGR-1 was obtained successfully by in vitro expression in BL21(DE3)cells.The Anti-rhDNGR-1 antibody(rabbit serum against CTLD of rhesus macaque DNGR-1)weas raised and could stain DNGR-1+ DCs in tissues.The recombinant CTLD was biologically active and could bind to apoptotic and dead cells in blood and cell line cells,and could also bind to pyroptotic cells induced by PMA and LPS.HeLa cells expressing recombinant rhesus macaque DNGR-1 could bind to apoptotic MT-4 cells and PBMC induced by freezing-thawing.3.Rhesus macaque DNGR-1mRNA levels were detected in all the tissues examined,with the highest expression levels in lymph nodes,spleen and blood,higher expression levels in gastrointestinal tissues,lung and tonsil.The DNGR-1mRNA levels were the lowest in skin,muscle,brain(cerebrum,cerebellum and spinal cord),the cardiovascular system(heart,artery),trachea and bone marrow.DNGR-1+DCs were found in intestinal lamina propria and in the junction area of cortex and medulla in inguinal lymph node.DNGR-1+DCs were also observed in white pulp boundary,junction of red and white pulp and marginal zone of spleen.However,DNGR-1+DCs were not found in the thymus and stomach fundic examined in this study.4.Alteration of DNGR-1 expression in the blood and tissues of immunodeficiency virus infected rhesus macaques was observed.DNGR-1mRNA levels in the blood of infected rhesus macaques were altered with time.DNGR-1mRNA levels on day 41 after inoculation with SHIV-sfl62p4(via rectum),on day 4,11,98 after inoculation with SHIV-sfl62p4(intravenously)and on day 4,11,182 after inoculation with SIVmac251(intravenously)were significantly decreased compared with that of normal animals.After 75 weeks,the expression levels were close to the normal level.During the early period of the second and third infection,the lowest levels of DNGR-1mRNA were observed and then they elevated to normal levels and reduced again during the process of infection.Before the end of third infection,DNGR-1mRNA levels raised to normal level again.The expression of DNGR-1 had an increasing trend in the gastrointestinal tissues,lymph nodes and blood.Notably,DNGR-1mRNA levels were significantly increased in the colon proximal and mesenteric lymph node.DNGR-1mRNA level in the blood was significantly decreased after HIV infection(3 years after infection).Furthermore,the expression levels of DNGR-1 in some infected humans may be affected by antiretroviral therapy(ART)and had no relationship with drug resistance.Negative correlation may be occurred between DNGR-1mRNA levels and virus load and CD4+T counts cells in some infected humans.5.Recombinant CTLD of rhesus macaque DNGR-1 could bind to apoptotic and dead blood cells from simian/human immunodeficiency virus infected rhesus macaques and humans.DNGR-1 binding cells were decreased in SIV infected rhesus macaques,while increased in ART treated AIDS patients.The percentage of recombinant CTLD protein binding apoptotic and dead cells and binding ability depend on the proportion and stages of apoptotic cells after infection.ConclusionsIn this study,a 1270bp sequence of rhesus macaque DNGR-1 cDNA was obtained.The encoding region of DNGR-1 was highly similar to that of humans.Rhesus macaque DNGR-1 shared similar antigenicity with human DNGR-1.The DNGR-1 expressed in almost all the tissues and the highest expression level was in lymph nodes,spleen and thymus.DNGR-1+DCs were found in intestinal lamina propria and in the junction area of cortex and medulla of inguinal lymph node.The recombinant CTLD of rhesus macaque DNGR-1 had biological activity and could bind to apoptotic,dead and pyroptotic cells.The DNGR-1mRNA levels were affected by immunodeficiency virus infection and identified the trends of change at different stages after infection.DNGR-1 binding cells were decreased in SIV infected rhesus macaques,while increased in ART treated AIDS patients.The percentage of recombinant CTLD protein binding apoptotic,dead cells and binding ability depend on proportion and different stages of apoptotic cells after infection.This study indicated that rhesus macaque DNGR-1 was highly similar to that of humans.Findings from rhesus macaque could be more easily translated into prevention,treatment and clinical application in AIDS and tumor diseases.The expression levels of DNGR-1 were significantly affected after immunodeficiency virus infection and ART failed to restore the normal levels,in which should be taken into consideration AIDS treatement and vaccine studies.