Sepsis-associated encephalopathy (SAE) is a severe and common neurological complication of sepsis, characterized by symptoms ranging from mild confusion, delirium, deep coma, and severe cognitive dysfunction. Previous epidemiological and bioinformatics studies have revealed that HLA DP and DRA molecule play a pivotal role during sepsis. However, the mechanism by which these class II molecule contribute to cognitive impairment in SAE remains unclear. using the peritoneal contamination and infection model (PCI) model in humanized transgenic HLA-DP401/DRA-IAβ-/- genotypes mice, we aimed to investigate the effects of HLA class II haplotypes/alleles on sepsis and elucidate the underlying mechanism leading to cognitive impairment. Our results indicated that the introduction of HLA DP/DRA molecule significantly increased mortality, exacerbated clinical symptoms, and elevated inflammatory cytokine responses in both serum and hippocampal tissue of septic mice. Cecal slurry (CS) injection induced robust microglia activation and severe pathological damage of hippocampus. Furthermore, transcriptome analysis revealed numerous differentially expressed genes (DEGs) and prominent mitochondrial dysfunction in HLA-DP/DRA-IAβ-/- mice subjected to PCI. Notably, CS injection up-regulated AMPK-α phosphorylation in IAβ-/- mice but not in HLA DP/DRA-IAβ-/- mice. Consistently, sepsis induced persistent neurocognitive deficits and long-term anxiety-like behaviors in HLA DP/DRA-IAβ-/- PCI mice. In conclusion, these data provide direct evidence that HLA class II molecules modulate the host response to sepsis and highlight a critical role of HLA-DP/DRA in exacerbating the severity of systemic infection. The introduction of the HLA-DP and HLA-DRA genes synergistically upregulated systemic and hippocampal inflammatory cytokines, worsened clinical outcomes, impaired memory performance, and exacerbated long-term anxiety-like behaviors.

Bitter taste receptors (TAS2Rs), initially identified for chemosensory roles in the tongue, are expressed in extraoral tissues, including the airways. However, to date, it remains unclear whether bitter signaling is associated with susceptibility to bacterial infection in the lower airways and whether bitter signaling actually participates in the immune response in lung infection has yet to be genetically established. Here, we investigated the role of TAS2R signaling in Staphylococcus aureus-induced murine pneumonia via wild-type (WT) and several mutants (mTas2r104-/-/105-/-, mTas2r105-/-/114-/-, mTas2r104-/-/105-/-/114-/-, Gnat3-/- and Gnat3-/-mTas2r104-/-/105-/-) mice. Genetic disruption of TAS2Rs altered compensatory expression of other bitter receptors in the trachea and lungs, but did not affect immune cell composition in the lungs or thymus. Bitter receptor-deficient mice exhibited exacerbated pulmonary lesions at day 3 (D3) post-infection. Pulmonary infection significantly upregulated mTas2r105,106, 107, 108, 126, 136, 138 and Gnat3 in the lung. TAS2R signaling deficiency downregulated the expression of cytokines (e.g., IL-10, MIP-2) and antimicrobial peptides in the lungs and trachea, increased CD68+ macrophages in D3 lung tissues, amplified Ki67+ cell proliferation in alveolar and bronchiolar regions, and even impaired recovery from lung injury by day 14 (D14). Mechanistically, bitter taste pathway disruption dysregulated the mTOR pathway, reduced eNOS expression, and delayed resolution of pneumonia-induced injury. In summary, the current results collectively indicate that bitter taste signaling can modulate innate immune and inflammatory responses during S. aureus-induced lung infection.

Recently, the expression of bitter taste receptors and their downstream taste signaling cascade has been widely found outside the gustatory system, indicating important physiological functions of bitter taste receptors in various extraoral organs, including testis, but little is known about their functions in spermatogenesis. Here, we describe the localization and expression pattern of taste signaling transduction molecules in the testis. Genetic mutation of bitter signaling transduction molecules decreased the litter size, the IVF rate, and the diameter of seminiferous tubules and even resulted in empty seminiferous tubules. Transmission electron microscopy observations further revealed that overdeveloped acrosomes adhered to atrophic round spermatids in double-mutant mice. Mutant mice lacking bitter taste receptor signaling exhibited a dysfunction of adenosine 5'-monophosphate-activated protein kinase and inducible nitric oxide synthase signaling pathways, and a decreased expression of zonula occludens-1 and β-catenin in testis, indicating a disruption for the structure and functions of the blood-testis barrier. Transcriptome analysis further showed that bitter taste signaling deficiency can alter the expression profile of transcripts related to signal pathways, hormone synthesis, cell adhesion molecules, the chemokine signaling pathway, and cell metabolism in the testis, which finally contribute to impaired male fertility. In short, our work provides previously unidentified in vivo evidence that bitter taste signaling plays a critical role in the maintenance of normal spermatogenesis. These data further support the concept that bitter taste receptors exert functions outside the gustatory system and may have implications for the diagnosis and management of human male infertility.

BACKGROUND:Staphylococcus aureus can cause serious infections by secreting many superantigen exotoxins in "carrier" or "pathogenic" states. HLA DQ and HLA DR humanized mice have been used as a small animal model to study the role of two molecules during S. aureus infection. However, the contribution of HLA DP to S. aureus infection is unknown yet. METHODS:In this study, we have produced HLA DP401 and HLA DRA0101 humanized mice by microinjection of C57BL/6J zygotes. Neo-floxed IAβ+/- mice were crossbred with Ella-Cre and further crossbred with HLA DP401 or HLA-DRA0101 humanized mice. After several rounds of traditional crossbreeding, we finally obtained HLA DP401-IAβ-/- and HLA DRA-IAβ-/- humanized mice, in which human DP401 or DRA0101 molecule was introduced into IAβ-/- mice deficient in endogenous murine MHC class II molecules. A transnasal infection murine model of S. aureus pneumonia was induced in the humanized mice by administering 2 × 108  CFU of S. aureus Newman dropwise into the nasal cavity. The immune responses and histopathology changes were further assessed in lungs in these infected mice. RESULTS:We evaluated the local and systemic effects of S. aureus delivered intranasally in HLA DP401-IAβ-/- and HLA DRA-IAβ-/- transgenic mice. S. aureus Newman infection significantly increased the mRNA level of IL 12p40 in lungs in humanized mice. An increase in IFN-γ and IL-6 protein was observed in HLA DRA-IAβ-/- mice. We observed a declining trend in the percentage of F4/80+ macrophages in lungs in HLA DP401-IAβ-/- mice and a decreasing ratio of CD4+ to CD8+ T cells in lungs in IAβ-/- mice and HLA DP401-IAβ-/- mice. A decreasing ratio of Vβ3+ to Vβ8+ T cells was also found in the lymph node of IAβ-/- mice and HLA DP401-IAβ-/- mice. S. aureus Newman infection resulted in a weaker pathological injury in lungs in IAβ-/- genetic background mice. CONCLUSION:These humanized mice will be an invaluable mouse model to resolve the pathological mechanism of S. aureus pneumonia and study what role DP molecule plays in S. aureus infection.

Background Bitter taste receptors (Tas2rs) are generally considered to sense various bitter compounds to escape the intake of toxic substances. Bitter taste receptors have been found to widely express in extraoral tissues and have important physiological functions outside the gustatory system in vivo.Methods To investigate the physiological functions of the bitter taste receptor cluster Tas2r106/Tas2r104/Tas2r105/Tas2r114 in lingual and extraoral tissues, multiple Tas2rs mutant mice and Gnat3 were produced using CRISPR/Cas9 gene-editing technique. A mixture containing Cas9 and sgRNA mRNAs for Tas2rs and Gnat3 gene was microinjected into the cytoplasm of the zygotes. Then, T7EN1 assays and sequencing were used to screen genetic mutation at the target sites in founder mice. Quantitative real-time polymerase chain reaction (qRT-PCR) and immunostaining were used to study the expression level of taste signaling cascade and bitter taste receptor in taste buds. Perception to taste substance was also studied using two-bottle preference tests.Results We successfully produced several Tas2rs and Gnat3 mutant mice using the CRISPR/Cas9 technique. Immunostaining results showed that the expression of GNAT3 and PLCB2 was not altered in Tas2rs mutant mice. But qRT-PCR results revealed the changed expression profile of mTas2rs gene in taste buds of these mutant mice. With two-bottle preference tests, these mutant mice eliminate responses to cycloheximide due to genetic mutation of Tas2r105. In addition, these mutant mice showed a loss of taste perception to quinine dihydrochloride, denatonium benzoate, and cucurbitacin B (CuB). Gnat3-mediated taste receptor and its signal pathway contribute to CuB perception.Conclusions These findings implied that these mutant mice would be a valuable means to understand the biological functions of TAS2Rs in extraoral tissues and investigate bitter compound-induced responses mediated by these TAS2Rs in many extraoral tissues.

目的 期望获得含有全长hACE2基因序列的hACE2人源化小鼠模型,为心血管疾病、新冠肺炎发病机制研究、药物与疫苗研发提供重要工具.方法 将纯化含有hACE2完整编码序列的BAC质粒,利用小鼠受精卵显微注射和输卵管移植技术获得多个首建鼠,分别育种建系,通过杂交育种获得稳定遗传F2代小鼠品系,进一步对F2代小鼠hACE2基因整合、基因拷贝数、相对荧光定量PCR、Western Blot和免疫荧光进行分析.结果 育种获得hACE2-6-9、hACE2-14-3、hACE2-15-1和hACE2-15-2共4个小鼠品系.基因整合结果显示,hACE2-6-9、hACE2-15-1和hACE2-15-2小鼠品系含有完整的hACE2基因座位点及较长的基因调控区,hACE2-14-3含有完整的hACE2基因座位点及3'UTR较短的基因调控区.相对荧光定量PCR和Western Blot检测结果显示,hACE2-6-9、hACE2-15-1和hACE2-15-2品系小鼠肠道中hACE2 mRNA和蛋白表达水平较低,而整合较短调控序列的hACE2-14-3小鼠品系则表达水平较高.免疫荧光染色结果显示hACE2-15-1品系小鼠肾小管和肺血管内皮细胞中均有hACE2表达.结论 获得了含有全长基因序列的hACE2人源化BAC转基因小鼠,保留完整的hACE2启动子及基因调控区,从而为心血管疾病、新冠肺炎发病机制研究、hACE2基因表达调控机制研究和药物、疫苗的研发提供重要工具.

Background : There are remarkable genetic differences between animal major histocompatibility complex（MHC） systems and the human leukocyte antigen（HLA） system. HLA transgenic humanized mouse model systems offer a much better method to study the HLA-A-related principal mechanisms for vaccine development and HLA-Arestricted responses against infection in human. Methods : A recombinant gene encoding the chimeric HLA-A30 monochain was constructed. This HHD molecule contains the following: α1-α2 domains of HLA-A30, α3 and cytoplasmic domains of H-2D b , linked at its N-terminus to the C-terminus of human β2m by a 15-amino-acid peptide linker. The recombinant gene encoding the chimeric HLA-A30 monochain cassette was introduced into bacterial artificial chromosome（BAC） CH502-67J3 containing the HLA-A01 gene locus by Red-mediated homologous recombination. Modified BAC CH502-67J3 was microinjected into the pronuclei of wild-type mouse oocytes. This humanized mouse model was further used to assess the immune responses against influenza A virus（H1N1） pdm09 clinically isolated from human patients. Immune cell population, cytokine production, and histopathology in the lung were analyzed. Results : We describe a novel human β2m-HLA-A30（α1α2）-H-2D b （α3 transmembrane cytoplasmic）（HHD） monochain transgenic mouse strain, which contains the intact HLA-A01 gene locus including 49 kb 5’-UTR and 74 kb 3’-UTR of HLA-A01*01. Five transgenic lines integrated into the large genomic region of HLA-A gene locus were obtained, and the robust expression of exogenous transgene was detected in various tissues from A30-18# and A30-19# lines encompassing the intact flanking sequences. Flow cytometry revealed that the introduction of a large genomic region in HLA-A gene locus can influence the immune cell constitution in humanized mice. Pdm09 infection caused a similar immune response among HLA-A30 Tg humanized mice and wild-type mice, and induced the rapid increase of cytokines, including IFN-γ, TNF-α, and IL-6, in both HLA-A30 humanized Tg mice and wild-type mice. The expression of HLA-A30 transgene was dramatically promoted in tissues from A30-9# line at 3 days post-infection（dpi）. Conclusions : We established a promising preclinical research animal model of HLA-A30 Tg humanized mouse, which could accelerate the identification of novel HLA-A30-restricted epitopes and vaccine development, and support the study of HLA-A-restricted responses against infection in humans.

Transient receptor potential vanillic acid 2 (TRPV2) are well recognized for their contributions to neuronal development, cardiac function, immunity and cancer. However, the precise roles for this thermo TRPchannels in neurological disorder remain unknown. In this study, we employed the CRISPR/Cas9 system to generate genetic mutations of TRPV2. Genetic mutation of TRPV2 resulted in autistic-like phenotypes in mice accompanied with the disordered electrical signals recorded by multi-channels in vivo. To determine possible molecular mechanisms, western blotting was further used to assess the possible involvement of several autism-related proteins. The significantly decreased expression of the R2 subunit of the GABA-B receptor in the hippocampus was observed. Together, our findings suggest that genetic mutation of TRPV2 induces autism-like behavior, results in decreased expression of the R2 subunit of the GABA-B receptor.

AbstractBackgroundHuman leukocyte antigen (HLA)‐DP is much less studied than other HLA class II antigens, that is, HLA‐DR and HLA‐DQ, etc. However, the accumulating data have suggested the important roles of DP‐restricted responses in the context of cancer, allergy, and infectious disease. Lack of animal models expressing these genes as authentic cis‐haplotypes blocks our understanding for the role of HLA‐DP haplotypes in immunity.MethodsTo explore the potential cis‐acting control elements involved in the transcriptional regulation of the HLA‐DPA1/DPB1 gene, we performed the expression analysis using bacterial artificial chromosome (BAC)‐based transgenic humanized mice in the C57BL/6 background, which carried the entire HLA‐DP401 gene locus. We further developed a mouse model of Staphylococcus aureus pneumonia in HLA‐DP401 humanized transgenic mice, and performed the analysis on the expression pattern of HLA‐DP401 and immunological responses in the model.ResultsIn this study, we screened and identified a BAC clone spanning the entire HLA‐DP gene locus. DNA from this clone was analyzed for integrity by pulsed‐field gel electrophoresis and then microinjected into fertilized mouse oocytes to produce transgenic founder animals. Nine sets of PCR primers for regional markers with an average distance of 15 kb between each primer were used to confirm the integrity of the transgene in the five transgenic lines carrying the HLA‐DPA1/DPB1 gene. Transgene copy numbers were determined by real‐time PCR analysis. HLA‐DP401 gene expression was analyzed at the mRNA and protein level. Although infection with S aureus Newman did not alter the percentage of immune cells in the spleen and thymus from the HLA‐DP401‐H2‐Aβ1 humanized mice. Increased expression of HLA‐DP401 was observed in the thymus of the humanized mice infected by S aureus.ConclusionsWe generated several BAC transgenic mice, and analyzed the expression of HLA‐DPA1/DPB1 in those mice. A model of S aureus‐induced pneumonia in the HLA‐DP401‐H2‐Aβ1−/− humanized mice was further developed, and S aureus infection upregulated the HLA‐DP401 expression in thymus of those humanized mice. These findings demonstrate the potential of those HLA‐DPA1/DPB1 transgenic humanized mice for developing animal models of infectious diseases and MHC‐associated immunological diseases.

T cells play a critical role in coronavirus diseases. How they do so in COVID-19 may be revealed by analyzing the epigenetic chromatin accessibility of cis- and trans-regulatory elements and creating transcriptomic immune profiles. We performed single-cell assay for transposase-accessible chromatin (scATAC) and single-cell RNA (scRNA) sequencing (seq) on the peripheral blood mononuclear cells (PBMCs) of severely ill/critical patients (SCPs) infected with COVID-19, moderate patients (MPs), and healthy volunteer controls (HCs). About 76,570 and 107,862 single cells were used, respectively, for analyzing the characteristics of chromatin accessibility and transcriptomic immune profiles by the application of scATAC-seq (nine cases) and scRNA-seq (15 cases). The scATAC-seq detected 28,535 different peaks in the three groups; among these peaks, 41.6 and 10.7% were located in the promoter and enhancer regions, respectively. Compared to HCs, among the peak-located genes in the total T cells and its subsets, CD4+ T and CD8+ T cells, from SCPs and MPs were enriched with inflammatory pathways, such as mitogen-activated protein kinase (MAPK) signaling pathway and tumor necrosis factor (TNF) signaling pathway. The motifs of TBX21 were less accessible in the CD4+ T cells of SCPs compared with those in MPs. Furthermore, the scRNA-seq showed that the proportion of T cells, especially the CD4+ T cells, was decreased in SCPs and MPs compared with those in HCs. Transcriptomic results revealed that histone-related genes, and inflammatory genes, such as NFKBIA, S100A9, and PIK3R1, were highly expressed in the total T cells, CD4+ T and CD8+ T cells, both in the cases of SCPs and MPs. In the CD4+ T cells, decreased T helper-1 (Th1) cells were observed in SCPs and MPs. In the CD8+T cells, activation markers, such as CD69 and HLA class II genes (HLA-DRA, HLA-DRB1, and HLA-DRB5), were significantly upregulated in SCPs. An integrated analysis of the data from scATAC-seq and scRNA-seq showed some consistency between the approaches. Cumulatively, we have generated a landscape of chromatin epigenetic status and transcriptomic immune profiles of T cells in patients with COVID-19. This has provided a deeper dissection of the characteristics of the T cells involved at a higher resolution than from previously obtained data merely by the scRNA-seq analysis. Our data led us to suggest that the T-cell inflammatory states accompanied with defective functions in the CD4+ T cells of SCPs may be the key factors for determining the pathogenesis of and recovery from COVID-19.

TAS1R taste receptors and their associated heterotrimeric G protein gustducin are strongly expressed in testis and sperm, but their functions and distribution in these tissues were unknown. Using transgenic mouse models, we show that taste signal transduction cascades (mTas1r3-Gnat3-Trmp5) are observed in testis form GFP transgenic mice. It is mTas1rs and mTas2rs, not Gnat3, that was expressed in leydig and sertoli cells. The pattern of mTas1r3 expression was different from that of mTas2r105 expression in seminiferous epithelium. Analysis of the seminiferous epithelium cycle show that both mTas1r3 and mTas2r105 is expressed in the spermatid stage, but mTas2r5 expression is found in spermatocyte stage. Conditional deletion of mTas1r3+ cells leads to male infertility, but do not affect the expression of taste signal transduction cascade during the spermatogenesis. The current results indicate a critical role for mTas1r3+ cell in sperm development and maturation.

目的 建立一种高效的C57BL/6J背景基因修饰小鼠的精子冷冻及体外受精(IVF)方法.方法 以C57BL/6J小鼠为研究对象,围绕冷冻保护液、冷冻精子浓度、复苏后精子处理方法及IVF培养液等方面进行研究,IVF后2-细胞发育率作为冷冻和复苏效率评价标准,对比各组之间发育情况.结果 以R18S3(含质量比为18％棉子糖和3％脱脂奶粉)、M-R18S3冷冻保护液[含终浓度为477 μmol/L硫代甘油(MTG)的R18S3冷冻液]进行精子冷冻,复苏后在HTF培养液中进行IVF,2-细胞发育率分别为8.4％和20.6％;以R18S3和M-R18S3作为冷冻保护液,以含浓度为1 mmol/L还原性谷胱甘肽(GSH)的HTF培养液进行IVF,2-细胞发育率分别为25.0％和43.5％.高浓度精子冷冻及复苏法可获得64.2％的2-细胞发育率;选取4种C57BL/6J背景基因修饰小鼠进行精子冷冻及IVF,2-细胞发育率34％～90％,胚胎移植后出生率40％～57％.结论 M-R18S3作为冷冻保护液,采用高浓度精子冷冻法进行精子冷冻,冷冻精子复苏后进行受精滴洗涤筛选,以含1 mmol/LGSH的HTF培养液进行冷冻精子IVF,此技术体系可以作为C57BL/6J背景基因修饰鼠的保种及品系恢复手段.

Type 2 taste receptors (TAS2Rs), a large family of GPCRs, were first discovered in the gustatory system, and are co-expressed in a subset of taste receptor cells and detect bitter-tasting compounds. Type 1 taste receptors (TAS1R3) function as an obligate partner for both the umami receptor and the sweet receptor. Recently, it has become clear that taste receptors are also expressed outside the gustatory system. Here, with Tas2r5-Cre/GFP and Tas1r3-Cre/GFP transgenic mice, the expression of taste receptors (Tas1r3 or Tas2r5) is observed in serous gland and mucous gland of tongue. Taste signal transduction cascade (Gnat3 and PLC-β2) is also detected in serous gland and mucous gland of tongue. After DTA expression in Tas2r5+ and Tas1r3+ cells, the expression of taste receptors and taste signal transduction cascade is ablated in serous and mucous gland of tongue.

Background: Lingual epithelia in the tongue tip are among the most rapidly regenerating tissues, but the mechanism of cell genesis in this tissue is still unknown. Previous study has suggested the existence of multiple stem cell pools in lingual epithelia and papillae. Like K14+ and Sox2+ cells, NTPDase2+ cells have characteristics of stem cells.Methods: We employed a system using doxycycline to conditionally ablate NTPDase2+ cells in lingual epithelia and papillae by regulated expression of the diphtheria toxin A（DTA） gene. Transgenic lines, which expressed the rtTA gene in NTPDase2+ cells, were produced by pronuclear injection of zygotes from C57 BL/6 mice using the BAC clone RP23-47 P18. The NTPDase2-rtTA transgenic mice were crossed with the TetO-DTA transgenic animals. The double transgenic mice were treated with doxycycline. Doxycycline（Dox） was diluted in 5% sucrose in water to a final concentration of 0.3-0.5 mg/mL and supplied as drinking water.Results: After 15 days of Dox induction, the expression of NTPDase2, Sox2 and K14 was ablated from lingual epithelia. DTA expression in NTPDase2+cells did not inhibit the turnover of GNAT3+ or PLCb2+ cells in taste buds,nor the expression of S100 b beneath lingual epithelia and papillae. After35 days ablation of NTPDase2+ cells, the basic structure of lingual epithelia and papillae remained intact. However, the ratio of cell to total tissue area was decreased in lingual epithelia and circumvallate（CV） papillae. DTA expression also inhibited the regeneration of filiform papillae on the dorsal surface of the tongue tip.Conclusions: These studies provide important insights into the understanding of dynamic equilibrium among the multiple stem cell populations present in the lingual epithelia and papillae.