Understanding global changes of the mouse brain proteome after vaginal infection with HSV-2 using a label-free shotgun approach.

Herpes simplex virus type 2 (HSV-2) is a common human pathogen that establishes lifelong latency in neurons of the nervous system. The number of severe central nervous system infections caused by the virus has increased recently. However, the pathogenesis of HSV-2 infection in the nervous system is not fully understood. Here, we demonstrated global proteomic changes in the brain tissue in BALB/c mice vaginally infected with HSV-2. Data are available via ProteomeXchange with identifier PXD034186. A total of 249 differentially expressed proteins were identified in infected brain tissue. The GO and KEGG enrichment analysis of these proteins indicated that they were mainly involved in the regulation of synapse formation and synaptic excitability. In addition, genes affecting autophagy, the development of other neurodegenerative diseases, and signaling pathways relevant to other neurologic diseases were identified. Additional experiments, comparing the brain tissue of asymptomatic and symptomatic mice showed a differential expression of proteins involved in synapse formation and synaptic transmission. Others were involved in autophagy, addiction, and signaling pathways of other neurologic diseases. These results suggest that changes in synaptic structure and function, as well as autophagy, may be related to the development of neurologic abnormalities that follow HSV-2 infection. We also identified a protein GluN2A encoded by Grin2a was continuously expressed at high levels after infection. We propose that GluN2A may be a key molecule in the pathogenesis of HSV-2-induced neurologic diseases.

HSV-1 Infection of Epithelial Dendritic Cells Is a Critical Strategy for Interfering with Antiviral Immunity.

Herpes simplex virus type 1 (HSV-1), an α subgroup member of the human herpesvirus family, infects cells via the binding of its various envelope glycoproteins to cellular membrane receptors, one of which is herpes virus entry mediator (HVEM), expressed on dendritic cells. Here, HVEM gene-deficient mice were used to investigate the immunologic effect elicited by the HSV-1 infection of dendritic cells. Dendritic cells expressing the surface marker CD11c showed an abnormal biological phenotype, including the altered transcription of various immune signaling molecules and inflammatory factors associated with innate immunity after viral replication. Furthermore, the viral infection of dendritic cells interfered with dendritic cell function in the lymph nodes, where these cells normally play roles in activating the T-cell response. Additionally, the mild clinicopathological manifestations observed during the acute phase of HSV-1 infection were associated with viral replication in dendritic cells.

The Mutation of the Genes Related to Neurovirulence in HSV-2 Produces an Attenuated Phenotype in Mice.

HSV-2 (Herpes simplex virus type 2) is a critical viral agent that mainly causes genital herpes and life-long latent infection in the dorsal root ganglia. Gene modification via CRISPR/Cas9 Clustered regularly interspaced short palindromic repeat sequences/CRISPR associated 9) was used here to construct HSV-2 mutant strains through the deletion of fragments of the RL1 (Repeat Long element 1) and/or LAT (Latency-associated Transcript) genes. The HSV-2 mutant strains LAT-HSV-2 and RL1-LAT-HSV-2 present different biological properties. The proliferation of RL1-LAT-HSV-2 in nerve cells was decreased significantly, and the plaques induced by RL1-LAT-HSV-2 in Vero cells were smaller than those induced by LAT-HSV-2 mutant and wild-type strains. The observation of mice infected with these two mutants compared to mice infected with the wild-type strain indicated that the mutant RL1-LAT-HSV-2 has an attenuated phenotype with reduced pathogenicity during both acute and latent infections and induces a stronger specific immune response than the wild-type strain, whereas the attenuation effect was not found in mice infected with the LAT-HSV-2 mutant containing the LAT gene deletion. However, the simultaneous mutation of both the RL1 and LAT genes did not completely restrict viral proliferation in nerve cells, indicating that multiple HSV genes are involved in viral replication in the neural system. This work suggests that the HSV-2 genes RL1 and/or LAT might be involved in the virulence mechanisms in mouse infections.

[Sorting and identification of human immature Langerhans cells].

Objective To isolate the immature Langerhans cells(LCs) of human epidermis and to identify the phenotype based on their surface marker. Methods After treatment of piecemeal tissues of human foreskin with dispase I digesting overnight, the separated epidermis was further digested by collagenaseIat 37DegreesCelsius in a shaker for 2 hours to produce a suspension of dispersive epithelium, in which, LCs population of CD3-CD14-CD1a+CD45+ was harvested in flow cytometry influx with certain antibodies and identified further by immunofluorescence assay of anti-CD207 antibody as while the vitality of these cells were observed with trypan blue staining. Results The population containing 91.31% of immature LCs was sorted and harvested, which showed 93.16% of survival rate. Conclusion Technical procedure of sorting immature LCs from human foreskin was established and ascertained being effective.

The microbiota in the intestinal and respiratory tracts of naked mole-rats revealed by high-throughput sequencing.

BACKGROUND: The naked mole-rat (NMR, Heterocephalus glaber) is being bred as a novel laboratory animal due to its unique biological characteristics, including longevity, cancer resistance, hypoxia tolerance, and pain insensitivity. It is expected that differences exist between the microbiota of wild NMRs and that of NMRs in an artificial environment. Overall, the effect of environment on changes in the NMR microbiota remains unknown. In an attempt to understand the microbiota composition of NMRs in captivity, variability in the microbiota of the intestinal and respiratory tracts of two groups of NMRs was assessed under two conditions. RESULTS: The results obtained by high-throughput sequencing revealed significant differences at the phylum, class, order, family and genus levels in the microbiota between the two groups of NMRs examined (first group in conventional environment, second group in barrier environment). For the trachea, 24 phyla and 533 genera and 26 phyla and 733 genera were identified for the first and second groups of animals. Regarding the cecum, 23 phyla and 385 genera and 25 phyla and 110 genera were identified in the microbiota of first and second groups of animals. There were no obvious differences between females and males or young and adult animals. CONCLUSIONS: Our results suggest that the intestinal and respiratory tract NMR microbiota changed during captivity, which may be related to the transition to the breeding environment. Such changes in the microbiota of NMRs may have an effect on the original characteristics, which may be the direction of further research studies.

Comparative study of macrophages in naked mole rats and ICR mice.

The domestic and foreign scholars have studied naked mole rats more focused on the respect such as its long life, resistant to low oxygen, little spontaneous tumor, but the study of the immune system is little. In this study, we compared the anatomy and tissue morphology of NMR and ICR mouse spleens and found that the gross appearance of the NNMR spleen differed from ICR. There were more macrophages in NNMR spleens than in ICR spleens. Furthermore, we focused on the differences of macrophages. We compared their phagocytic capabilities and the data showed that NNMR macrophages are more phagocytic than ICR mouse macrophages. We also used polyI:C and LPS to stimulate the NMR and ICR macrophages and then measured the immune response as expression of certain TLR signaling molecules. After stimulation, there was a lower increase in apoptosis of NMR macrophages than ICR macrophages and a non-significant increased expression of TLRs in NMR macrophages than in ICR macrophages. In contrast, NF-κB proteins increased more significantly in NMR's than in ICR's and the expression of downstream cytokines in NMR macrophages also increased more than in ICR macrophages. Based on these results, we hypothesize that in addition to being able to eat foreign matter, NMR macrophages can activate the TLRs, start the NF-κB and produce a large number of cytokines to enhance immune response, so as to protect the body from outside interference when the virus or bacteria invading.

Levels of Leydig cell autophagy regulate the fertility of male naked mole-rats.

Fertility is abolished in nonbreeding males in colonies of natal naked mole-rats (NMRs). Although spermatogenesis occurs in both breeding and nonbreeding male NMRs, the mechanisms underlying the differences in fertility between breeders and nonbreeders remain unexplored. In this study, a significant decrease in autophagy was observed in Leydig cells of the testis from nonbreeding male NMRs. This alteration was visualised as a significant decrease in the levels of autophagy-related gene 7 (Atg7), Atg5, microtubule-associated protein 1A/B light chain 3 (LC3-II/I) and the number of autophagosomes and an increase in P62 levels using Western blotting analyses. Furthermore, monodansylcadaverine (MDC) staining and Western blot analyses revealed that testosterone production decreased in nonbreeding male NMR Leydig cells, this decrease was associated with a reduction in autophagy. Primary Leydig cells from breeding and nonbreeding male NMRs were processed to investigate the effect of an autophagy inhibitor (3-MA, 3-methyladenine) or an autophagy activator (rapamycin) on testosterone production. Rapamycin induced an increase in testosterone production in NMR Leydig cells, whereas 3-MA had the opposite effect. Consequently, spermatogenesis, the weight of the testis, and androgen levels were dramatically reduced in nonbreeding male NMRs. While rapamycin treatment restored the fertility of nonbreeding male NMRs. Based on these results, inadequate autophagy correlates with a decrease in steroid production in nonbreeding male NMR Leydig cells, which may ultimately influence the spermatogenesis and fertilities of these animals.

Transcriptome sequencing of the naked mole rat (Heterocephalus glaber) and identification of hypoxia tolerance genes.

The naked mole rat (NMR; Heterocephalus glaber) is a small rodent species found in regions of Kenya, Ethiopia and Somalia. It has a high tolerance for hypoxia and is thus considered one of the most important natural models for studying hypoxia tolerance mechanisms. The various mechanisms underlying the NMR's hypoxia tolerance are beginning to be understood at different levels of organization, and next-generation sequencing methods promise to expand this understanding to the level of gene expression. In this study, we examined the sequence and transcript abundance data of the muscle transcriptome of NMRs exposed to hypoxia using the Illumina HiSeq 2500 system to clarify the possible genomic adaptive responses to the hypoxic underground surroundings. The RNA-seq raw FastQ data were mapped against the NMR genome. We identified 2337 differentially expressed genes (DEGs) by comparison of the hypoxic and control groups. Functional annotation of the DEGs by gene ontology (GO) analysis revealed enrichment of hypoxia stress-related GO categories, including 'biological regulation', 'cellular process', 'ion transport' and 'cell-cell signaling'. Enrichment of DEGs in signaling pathways was analyzed against the Kyoto Encyclopedia of Genes and Genomes (KEGG) database to identify possible interactions between DEGs. The results revealed significant enrichment of DEGs in focal adhesion, the mitogen-activated protein kinase (MAPK) signaling pathway and the glycine, serine and threonine metabolism pathway. Furthermore, inhibition of DEGs (STMN1, MAPK8IP1 and MAPK10) expression induced apoptosis and arrested cell growth in NMR fibroblasts following hypoxia. Thus, this global transcriptome analysis of NMRs can provide an important genetic resource for the study of hypoxia tolerance in mammals. Furthermore, the identified DEGs may provide important molecular targets for biomedical research into therapeutic strategies for stroke and cardiovascular diseases.

HIF-1α contributes to hypoxia adaptation of the naked mole rat.

BACKGROUND/AIMS: Naked mole rats (NMRs) spend their lives in burrow systems containing very low levels of oxygen, indicating long-term hypoxic exposure, and suggesting that pathological changes caused by hypoxia are attenuated or absent in this hypoxia-tolerant species. The mechanisms underlying NMRs hypoxia tolerance remain poorly understood. In this study, we explored whether hypoxia inducible factor 1α (HIF-1α), and vascular endothelial growth factor A (VEGFA) play a role in NMRs adaption to hypoxia. METHODS: Primary hepatic stellate cells (HSCs) isolated from NMRs and mice were treated with 50 µM YC-1, 50 µM KC7F2 or VEGFA siRNA. HIF-1α or VEGFA expression was detected by Western blot and real-time PCR. Apoptosis was determined by flow cytometry. The expression of autophagy markers (LC3 and p62) was detected by Western blot. RESULTS: Our results showed that HIF-1α and VEGFA expression in NMRs was significantly higher than in hypoxia-sensitive mice. Inhibition of HIF-1α expression induced apoptosis in both NMR and mouse HSCs following hypoxia. However, blocking VEGFA transcription results in a significant increase of apoptosis in both NMR and mouse HSCs before and after hypoxia. In addition, NMR HSCs displayed higher levels of autophagy (ratio of LC3ΙΙ/LC3Ι = 9.6) than mouse HSCs (relative ratio of LC3ΙΙ/ LC3Ι = 4.9) under hypoxic conditions. CONCLUSION: We conclude that HIF-1α activation may be an important mechanism for hypoxia adaption. However, high expression of VEGFA follows HIF-1α activation in NMRs.

H2O2 treatment or serum deprivation induces autophagy and apoptosis in naked mole-rat skin fibroblasts by inhibiting the PI3K/Akt signaling pathway.

Naked mole-rats (NMR; Heterocephalus glaber) display extreme longevity and resistance to cancer. Here, we examined whether autophagy contributes to the longevity of NMRs by assessing the effects of the PI3K/Akt pathway inhibitor LY294002 and the autophagy inhibitor chloroquine (CQ) on autophagy and apoptosis in NMR skin fibroblasts. Serum starvation, H2O2 treatment, and LY294002 treatment all increased the LC3-II/LC3-I ratio and numbers of double-membraned autophagosomes and autophagic vacuoles, and decreased levels of p70S6K, p-AktSer473, and p-AktThr308. By contrast, CQ treatment decreased p70S6K, AktSer473, and AktThr308 levels. The Bax/Bcl-2 ratio increased after 12 h of exposure to LY294002 or CQ. These data show that inhibiting the Akt pathway promotes autophagy and apoptosis in NMR skin fibroblasts. Furthermore, LY294002 or CQ treatment decreased caspase-3, p53, and HIF1-α levels, suggesting that serum starvation or H2O2 treatment increase autophagy and apoptosis in NMR skin fibroblasts by inhibiting the PI3K/Akt pathway. CQ-induced inhibition of late autophagy stages also prevented Akt activation and induced apoptosis. Finally, the HIF-1α and p53 pathways were involved in serum starvation- or H2O2-induced autophagy in NMR skin fibroblasts.