Avian H7N9 influenza viruses cause sporadic outbreaks of human infections and threaten to cause a major pandemic. The breadth of B cell responses to natural infection and the dominant antigenic sites recognized during first exposure to H7 HA following infection are incompletely understood. Here, we studied the B cell response to H7 HA of 2 individuals who had recovered from natural H7N9 virus infection. We used competition binding, hydrogen-deuterium mass spectrometry, and single-particle negative stain electron microscopy to identify the patterns of molecular recognition of the antibody responses to H7 HA. We found that circulating H7-reactive B cells recognized a diverse antigenic landscape on the HA molecule, including HA head domain epitopes in antigenic sites A and B and in the trimer interface-II region and epitopes in the stem region. Most H7 antibodies exhibited little heterosubtypic breadth, but many recognized a wide diversity of unrelated H7 strains. We tested the antibodies for functional activity and identified clones with diverse patterns of inhibition, including neutralizing, hemagglutination- or egress-inhibiting, or HA trimer–disrupting activities. Thus, the human B cell response to primary H7 natural infection is diverse, highly functional, and broad for recognition of diverse H7 strains. Influenza A viruses (IAVs) are categorized based on their surface proteins, HA and neuraminidase (NA), into 16 and 9 subtypes, respectively . H7Nx viSubtype H7 avian origin IAVs paired with N2, N3, N4, N7, or N9 NAs have caused more than 1567 confirmed human infections since 2003 . PreviouWe obtained peripheral blood mononuclear cells (PBMCs) and blood samples from 2 individuals born before 1968, approximately 11 months after recovery from naturally occurring laboratory-confirmed H7N9 influenza virus infection acquired in China . To detehttps://doi.org/10.1172/jci.insight.152403DS1). All mAbs exhibited low mutation frequencies in their antibody variable gene sequences, consistent with the retention of clones in the memory B cell compartment that are typical of a primary immune response following first infection.PBMCs were transformed with Epstein-Barr virus and other B cell stimuli, as previously described , 29, andThe isolated anti-H7 mAbs bound in ELISA to the corresponding recombinant H7 HA protein of the infecting isolate  and to the closely related SH13 strain HA used in LCL screening . The majThe influenza HA protein mediates multiple functions in the virus life cycle, including binding the virus to sialic acid on the surface of target cells (attachment) and fusing the virus envelope with the late endosomal membrane. Anti-HA antibodies can interfere with virus replication at different stages in the life cycle of the influenza virus, such as by inhibiting receptor binding and membrane fusion or functions associated with the viral NA like egress and release . To deteTo determine the specific stage in the virus life cycle and the mechanism of inhibitory action for the neutralizing anti-H7 mAbs, we next performed HAI assays with the SH13 H7N9 IDCDC-RG32A virus. More than half (8 of 18) of mAbs exhibited HAI activity . Of noteWe next defined groups of mAbs that bind to common major antigenic sites using a competition-binding assay with recombinant trimeric SH13 HA ectodomain . The assNext, we conducted studies to confirm the inferred location of the sites corresponding to competition-binding groups with 2 complementary structural analyses: hydrogen-deuterium exchange mass spectrometry (HDX-MS) and negative stain electron microscopy (nsEM) of H7/mAb complexes . ResultsSerology studies have shown that human H7N9 virus infection stimulates the induction of H7-reactive antibody responses that persist for at least several years , 24, 38.100 values at approximately 5 ng/mL concentration  on the HA globular head  and antiInformation on epitopes targeted by broadly reactive neutralizing and/or protective mAbs is a primary requirement for structure-based vaccine antigen design. Isolation and characterization of naturally elicited H7-specific antibodies allow us to confirm the importance of previously characterized accessible, surface-exposed, and vulnerable sites of H7 HA — site A, site B, and the stem region — and reveal an additional area of vulnerability, the TI-II site. On the other hand, this type of study can reveal that some sites of vulnerability for antibodies that are potent in neutralization and/or protection can only be recognized by a restricted set of antibodies with unusual features, like large numbers of somatic mutations in the variable gene sequences and complementarity-determining regions (CDRs) or extra-long or -short CDR3 loops. Such constraints may make development of protective immunity after vaccination more challenging in most people. The findings here are interesting in that antibodies identified after H7N9 infection in our work have low mutation frequencies in the variable gene sequences, and there is no requirement for especially short or long heavy chain CDR3 loops. Together with the high prevalence of virtually pan-H7Nx, broadly reactive antibodies in the panel isolated from H7N9 survivors’ memory B cells, these 2 considerations suggest a solid foundation for designing structure-based vaccines against avian H7Nx viruses. At the same time, the lack of reactivity to heterosubtypic HAs beyond H15 HA exhibited by the mAbs isolated suggests that the head domain site A, site B, and TI-II epitopes  are not likely to be used successfully as the target for vaccine antigen design for universal influenza vaccine development.In conclusion, these studies show that mAbs induced by naturally occurring H7N9 infection in humans exhibit broad cross-reactivity within the H7 and H15 subtypes, target mainly antigenic site A and the TI-II site on the H7 head domain, and neutralize virus by diverse mechanisms. Given that the antibody variable genes for the mAbs isolated here contained few somatic mutations, the results suggest these antibodies mostly comprised a primary immune response to this H7N9 infection. These findings help inform the development of universal or avian H7N9 influenza vaccines and offer tools to characterize and evaluate new antigens and candidate vaccine viruses.The reverse-genetics–derived virus with the H7N9 HA and NA genes on a PR8 backbone, designated A/Shanghai/2/2013 (H7N9)-PR8-IDCDC-RG32A , was propagated in embryonated chicken eggs (Charles River Laboratories) and manipulated under Biosafety Level 2 (BSL-2) conditions with BSL-3 practices. Virus titration and neutralization assays were performed on MDCK  epithelial cells. Cells were determined to be mycoplasma free at laboratory passage 2 and monthly when in use.A cDNA encoding the HA genes from the IAVs A/Shanghai/02/2013, A/British Columbia/1/2015, A/Hunan/02650/2016, A/Guangdong/17SF003/2016, A/Hong Kong/125/2017 H7N9, A/England/268/1996 and A/Netherlands/219/2003 H7N7, A/Canada/rv504/2004 H7N3 and A/New York/107/2003 H7N2, A/shearwater/Western Australia/2576/1979 H15N9, A/Hong Kong/1/1968, A/California/07/2004, A/Switzerland/9715293/2013 H3N2, A/mallard duck/Astrakhan/263/1982 H14N5, A/duck/Czechoslovakia/1956 H4N6, A/Taiwan/2/2013 H6N1, A/Singapore/1/1957 H2N2, A/Vietnam/1203/2004 H5N1, A/Puerto Rico/8/1934, A/Texas/36/1991 and A/California/04/2009pnd H1N1, and A/Hong Kong/1073/1999 H9N2 were optimized for expression. The cDNAs were synthesized (GenScript) as soluble trimeric constructs by replacing the transmembrane and cytoplasmic domain sequences with cDNAs encoding the GCN4 trimerization domain and a His6-tag at the C-terminus. Synthesized genes were subcloned into the pcDNA3.1(+) mammalian expression vector (Thermo Fisher Scientific). HA protein was expressed by transient transfection of FreeStyle 293-F cells  with polyethyleneimine transfection reagent and was grown in expression medium . The supernatants were harvested after 7 days, filter-sterilized with a 0.4 μm filter, and purified with HisTrap TALON FF crude columns .Human hybridoma cell lines secreting anti-H7 mAbs were generated using methods as described previously , 29. BriFor the expression of recombinant rH7-243 and rH7-197 Fabs, the nucleotide sequences of antibody heavy and light chain antibody variable genes were codon optimized for mammalian expression and synthesized at Twist Biosciences. The resulting gene fragments were cloned directly at Twist Biosciences into the pTwist CMV BetaGlobin WPRE NEO mammalian expression vector (Twist Biosciences). MAb heavy/light chain constructs were used to transfect Expi293F cells  transiently, and supernatants were harvested after culturing for 6 to 7 days. Recombinant Fabs were purified with Anti-CH1 CaptureSelect column (Cytiva). IgGs produced by the corresponding human mAb-secreting hybridoma cell lines in serum-free medium  were purified by affinity chromatography using HiTrap MabSelect SuRe column .MAb IgGs were incubated 3 to 4 hours with 4% w/w papain at 37°C. Then 0.5 M iodoacetamide was added to stop digestion. CaptureSelect IgG-Fc multispecies resin (Thermo Fisher Scientific) was added to the digested material and incubated overnight at 4°C. The supernatant was separated from Fc-bound resin with a 0.2 μm filter and then purified over a Superdex 200 size exclusion column (Cytiva). The Fab peak was concentrated for complexing with antigen.50 values for mAbs were determined using Prism 5.0 software (GraphPad Software) after log transformation of antibody concentration using nonlinear regression analysis. Each experiment was conducted twice independently.For screening ELISA, plates were incubated with culture supernatants from transformed B cells. For cross-reactivity assays, serum samples were assessed at 3-fold dilutions starting from 1/20, in triplicate, and purified mAbs were assessed at concentrations ranging from 10 μg/mL to 0.1 ng/mL, in triplicate. We performed ELISAs using 384-well plates coated overnight at 1 μg/mL with the recombinant HA protein of interest. The plates then were blocked with 50 μL of 5% nonfat dry milk and 0.1% Tween-20 in D-PBS (ELISA buffer) for 1 hour at room temperature. The plates were washed and serial 3-fold dilutions of the serum samples or mAbs were added to the wells and incubated for an hour. The plates were washed and 25 μL of ELISA buffer containing a 1:4000 dilution of anti-human IgG alkaline phosphatase conjugate  was added. After a final wash, 25 μL of phosphatase substrate solution (1 mg/mL p-nitrophenol phosphate in 1 M Tris aminomethane) was added to the plates, then incubated for 1 hour, and the optical density values were measured at 405 nm wavelength on a BioTek plate reader. The plates were washed 3 times between each step with PBS containing 0.05% Tween-20. The area under the curve was calculated in Prism software (GraphPad Software). EC100 value was defined as the lowest antibody concentration that inhibited hemagglutination of red blood cells.The HAI assay was performed with A/Shanghai/2/2013 (H7N9)-PR8-IDCDC-RG32A. For HAI, 25 μL of 4 HA units of virus were incubated for 1 hour at room temperature with 25 μL 2-fold serial dilutions of antibodies starting at 10 μg/mL in PBS. The 50 μL of antibody-virus mixture was incubated for 45 minutes at 4°C with 50 μL of turkey red blood cells  diluted in PBS. The IC2. The cells were then washed with VGM again and replenished with VGM containing 3-fold serial dilutions of mAbs or zanamivir (GlaxoSmithKline), starting at the highest concentration of mAbs 10 μg/mL or equimolar. The plates were incubated for 21 hours at 37°C in 5% CO2, and the supernatants were collected for performing the HA assay. For HA assay, we used turkey red blood cells  that were washed and diluted to 0.5% in PBS. A volume of 50 μL of the supernatants was incubated with 50 μL of the 0.5% turkey red blood cells in V-bottom plates for 1 hour at 4°C. The IC100 values were defined as the lowest antibody concentration added to infected MDCK cells that corresponded to absence of virus in supernatant according to HA of red blood cells.MDCK cells were seeded in plain Dulbecco’s modified Eagle medium  containing 10% FBS in 96-well plates overnight. The cells were washed 3 times with Virus Growth Medium (VGM)  and 100 μL of 1 multiplicity of infection (MOI) of A/Shanghai/2/2013 (H7N9)-PR8-IDCDC-RG32A virus in VGM added to the cells and incubated for 3 hours at 37°C in 5% CO50 values were calculated using GraphPad Prism.To screen neutralizing activity of mAbs, we used a high-throughput and quantitative RTCA and xCELLigence Analyzer (ACEA Biosciences Inc.) that assesses kinetic changes in cell physiology, including virus-induced CPE. Fifty microliters of cell culture medium  was added to each well of a 96-well E-plate to obtain a background reading. Forty thousand  MDCK cells in 50 μL of cell culture medium were seeded per well, and the plate was placed on the analyzer. Measurements were taken automatically every 15 minutes, and the sensograms were visualized using RTCA software version 2.1.0 (ACEA Biosciences Inc). A/Shanghai/2/2013 (H7N9)-PR8-IDCDC-RG32A virus (0.25 MOI) was mixed with six 3-fold dilutions of mAbs starting at 10 mg/mL in a total volume of 100 μL and incubated for 1 hour at room temperature. At 22 to 24 hours after seeding the cells, the virus/mAb mixtures were added in 3 replicates to the cells in 96-well E-plates. Wells containing virus only in the absence of mAb and wells containing only MDCK cells in medium were included on each plate as controls. Plates were measured continuously (every 15 minutes) for over 48 hours to assess virus neutralization. Dose-dependent neutralization curves were graphed, and neutralization ICRecombinant rH7-243 and rH7-197 and cleaved from hybridoma-derived IgG H7-247, H7-236, and H7-238 Fabs were incubated with uncleaved H7 SH13 HA or H7 Hunan trimer overnight at 4 times molar excess of Fab. Then rH7-243 and rH7-247 were incubated with HA for 20 seconds to catch an intact complex before degradation. The complex was added to carbon-coated 400 mesh copper grids and stained with 2% uranyl formate. Micrographs were collected on a 120 kV Tecnai Spirit microscope with a 4kx4k TemCam F416 camera using Leginon. Images were processed within Appion , and 2D BLI on an Octet Red instrument (FortéBio) was used to perform competition-binding assays. We loaded the H7 HA from A/Shanghai/2/2013 H7N9 with 6xHis-tag onto Anti-Penta-HIS biosensors  at a concentration of 10 μg/mL, then tested binding of 2 successively applied mAbs at 50 μg/mL concentration. Antigen and mAb dilutions were made in 1X Kinetics Buffer . The mAbs were defined as competing if the first Ab reduced binding of the second Ab by more than 70%. The mAbs were defined as noncompeting if the first Ab reduced binding of the second by less than 30%.2O (pH 7.5), at 0°C, and then quenched with 0.8% formic acid (v/v) containing various concentrations of GuHCl (0.8 to 6.4 M) and Tris(2-carboxyethyl)phosphine (TCEP) (0.1 or 1.0 M). After incubating on ice for 5 minutes, the quenched samples were diluted 3-fold with 0.8% formic acid (v/v) containing 16.6% (v/v) glycerol and then were frozen at –80°C until they were transferred to the cryogenic autosampler. Using the quench buffer of 6.4 M GuHCl, 1 M TCEP in 0.8% formic acid, gave an optimal peptide coverage map. The samples later were thawed automatically on ice and then immediately passed over an AL-20-pepsin column . The resulting peptides were collected on a C18 trap and separated on a C18 reversed-phase column  running a linear gradient of 0.046% (v/v) trifluoroacetic acid, 6.4% (v/v) acetonitrile to 0.03% (v/v) trifluoroacetic acid, 38.4% (v/v) acetonitrile over 30 minutes with column effluent directed into an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific). Data were acquired in both data-dependent MS:MS mode and MS1 profile mode. Proteome Discoverer software (Thermo Fisher Scientific) was used to identify the sequence of the peptide ions. HDEXaminer software  was used for the analysis of the mass spectra. IgG-bound HAs were prepared by mixing IgGs with trimeric H7 HA at a 1:1.1 (HA/IgG) stoichiometric ratio. The mixtures were incubated at 25°C for 30 minutes. All functionally deuterated samples, except for the equilibrium-deuterated control, and buffers were prechilled on ice and prepared in the cold room. Functional deuterium-hydrogen exchange reaction was initiated by diluting free HA or antibody-bound HA stock solution with D2O buffer  at a 1:2 v/v ratio. At 10, 100, and 1000 seconds, the quench solution was added to the respective samples, and incubated on ice for 5 minutes, then diluted 3-fold with 0.8% formic acid before freezing at –80°C. In addition, nondeuterated samples, equilibrium-deuterated back-exchange control samples, were prepared as previously described  after log transformation of antibody concentration using nonlinear regression analysis. Each experiment was conducted twice independently. Ab IC50 values were calculated using a formula based on the Spearman-Kärber equation . For binding curves, data are shown as mean ± SD of assay triplicates. ECequation . The Pea Life v4 , which wA blood sample from 2 survivors of natural H7N9 infection, described in a case report previously , was colIMG, SB, ABW, RHC, and JEC planned experiments; IMG, SL, SB, NK, and HLT performed experiments; IMG and JEC wrote the manuscript; IMG, SB, NK, RGB, AT, SL, HLT, RHC, ABW, JEC reviewed, edited, and approved the final manuscript; and JEC and ABW obtained funding.


Sgpl1-knockout mice fail to develop germ cells, resulting in infertility. However, the molecular mechanism remains unclear. The results of the present study showed that SGPL1 was expressed mainly in granulosa cells, Leydig cells, spermatocytes, and round spermatids. Sgpl1 deletion led to S1P accumulation in the gonads. In the ovary, S1P decreased natriuretic peptide receptor 2 (NPR2) activity in granulosa cells and inhibited early follicle growth. In the testis, S1P increased the levels of cyclin-dependent kinase inhibitor 1A (p21) and apoptosis in Leydig cells, thus resulting in spermatogenesis arrest. These results indicate that Sgpl1 deletion increases intracellular S1P levels, resulting in the arrest of female and male germ cell development via different signaling pathways.Sphingosine phosphate lyase 1 (SGPL1) is a highly conserved enzyme that irreversibly degrades sphingosine-1-phosphate (S1P).  The follicle is the basic unit of the ovary. Follicular development initiates with the transition of the primordial stage to preantral follicles, and then these preantral follicles develop into large antral follicles under follicle-stimulating hormone (FSH) stimulation2. Natriuretic peptide type C  and its receptor natriuretic peptide receptor 2 (NPR2) maintain meiotic arrest of oocytes within full-grown follicles4. When luteinizing hormone (LH) surges occur, NPR2 guanylyl cyclase activity is decreased, and then oocytes resume meiosis6. On the other hand, NPPC/NPR2 is also essential for early follicle development. NPPC can promote early follicle growth by NPR2-produced cyclic guanosine monophosphate (cGMP)7, and Npr2-null mice exhibit small ovaries with very few antral follicles and infertility8. Morphogenesis of the testis begins shortly after birth and continues until puberty. Seminiferous tubules and Leydig cells are two key components of the testes. Seminiferous tubules are the site of spermatogenesis. Leydig cells secrete testosterone, which diffuses into the seminiferous tubules and promotes spermatogenesis. Mice with testosterone deficiency by LH receptor (Lhcgr) deletion or with androgen receptor (Ar) deletion exhibit spermatogenesis arrest10.Normal gonad development is crucial for the production of mature gametes and the secretion of sexual hormones11. The most severe patients suffer from hydrops fetalis and/or perinatal death. Male infant patients exhibit a small penis, no palpable testes, low baseline testosterone levels, and/or high gonadotrophins12. Consistent with the human disease phenotype, SGPL1 deficiency in mice leads to the absence of spermatids by decreasing Leydig cell populations and testosterone production13. On the other hand, Sgpl1 deletion also causes few antral follicles to develop in the ovary13. However, the molecular mechanisms of SGPL1 in germ cell development remain unclear. SGPL1 is an irreversible degradation enzyme that mediates cleavage of the lipid-signaling molecule sphingosine-1-phosphate (S1P)14. SGPL1 deficiency impairs its capacity to degrade S1P, resulting in high levels of S1P in the mouse liver15 and thymus16. In addition, the other two reversible S1P phosphatases, SGPP1 and SGPP2, can also regulate S1P levels17. S1P, a unique bioactive sphingolipid, is produced intracellularly by two sphingosine kinases: SphK1 and SphK2 (ref. 18). S1P plays a vital role in regulating cell proliferation, survival, movement, and calcium homeostasis. Extracellular S1P exerts its effects by binding to its specific G protein-coupled receptors (S1PRs) to regulate downstream signals20. S1P can also mobilize calcium release from the endoplasmic reticulum to mediate various cellular functions22. Our previous studies have indicated that S1P decreases NPR2 activity by elevating calcium levels in cumulus cells23. On the other hand, overexpression of SphK increases S1P levels and then enhances the transcription of p21 in human MCF-7 breast cancer cells24. p21 can block cell proliferation in the G1 phase26, and promote cell apoptosis in granulocytes27.In recent years, many clinical cases have been reported that the mutation in sphingosine phosphate lyase 1 (SGPL1) gene can result in nephrotic syndrome and hypogonadismSgpl1 elevated S1P levels. S1P accumulation decreased NPR2 activity to arrest follicle development in the ovaries and increased p21 expression and Leydig cell apoptosis to induce defects in spermatogenesis in the testes. Collectively, SGPL1 modulates S1P levels, which are crucial for germ cell development.This study focuses on the molecular mechanisms of SGPL1 in germ cell development. We found that deletion of Sgpl1 were significantly higher than those of Sgpp1 and Sgpp2  mice  was significantly increased and the binding affinity of NPR2 for NPPC (green color) was significantly decreased in granulosa cells of KO mice -, BrdU-, and Ki-67-positive granulosa cells in KO mice were significantly decreased compared with those in WT mice  led to a significant decrease in follicle size Fig. . FurtherNpr2 deletion had no effect on testis development8. Therefore, we studied the effect of Sgpl1 deletion on p21 expression. p21 was expressed in the nuclei of Leydig cells and spermatocytes  and cell growth genes  could potentially arrest granulosa cell proliferation. In addition, downregulation of tubulin genes  could potentially impair cellular structure and gap junction formation in granulosa cells28. On the other hand, downregulation of genes associated with glycolysis/gluconeogenesis , amino acid transport , and fatty acid metabolism  could potentially impair granulosa cell nutrition and energy metabolism. Thus, deletion of Sgpl1 could potentially impair the growth and metabolism of granulosa cells, which could participate in the arrest of follicle development.We applied RNA sequencing (RNA-seq) technology to further investigate the molecular mechanisms of SGPL1 in mouse germ cell development. In the ovary, a total of 858 transcripts, including 431 upregulated and 427 downregulated transcripts, were differentially expressed in granulosa cells of KO mice compared with those of WT mice Fig. . The chaism Fig. . For exaSgpl1 KO testes could impair testis development  and upregulation of cell apoptosis-related genes  could potentially increase the apoptosis of Leydig cells and spermatocytes. In addition, downregulation of genes associated with steroid hormone synthesis  could potentially impair testosterone production. On the other hand, downregulation of spermatogenesis-associated genes  and upregulation of genes associated with spermatogenesis suppression  could potentially impair spermatogenesis. Thus, these changed transcripts in Sgpl1 KO testes could participate in the arrest of spermatogenesis.In the testis, a total of 5426 transcripts, including 1263 upregulated and 4163 downregulated transcripts, were differentially expressed in KO mice compared with WT mice Fig. . Then, tent Fig. . These i14. Sgpl1 KO mice are unable to produce mature germ cells and exhibit infertility13. In the present study, Sgpl1 deletion led to the accumulation of S1P in the gonads. In the ovary, the increase in S1P decreased NPR2 activity in granulosa cells and inhibited early follicle development. In the testis, the increase in S1P promoted p21 expression and cell apoptosis in Leydig cells. The decrease in Leydig cell populations and testosterone levels leads to failure in spermatogenesis13. Therefore, SGPL1 plays a vital role in the development of germ cells by regulating S1P levels.SGPL1 is the key regulatory enzyme in the sphingolipid degradative pathway29. In our study, SGPL1 was the main S1P degradation enzyme in the gonads. Furthermore, deletion of Sgpl1 led to S1P accumulation in the ovaries and testes, consistent with a previous report that the inhibition of SGPL1 extremely increases S1P levels in the mouse liver15. Thus, SGPL1 plays an important role in regulating intracellular S1P levels in the gonads. S1P acts as a phospholipid signaling molecule that can mobilize intracellular calcium release from the endoplasmic reticulum23 and then decrease NPR2 activity by reducing its binding affinity for NPPC in cumulus cells and vascular smooth muscle cells30. Interestingly, deletion of Sgpl1 also led to calcium increase and NPR2 inactivity in granulosa cells. In vitro, S1P decreased the activity of NPR2 in granulosa cells by increasing intracellular calcium. On the other hand, S1P could directly decrease NPPC expression in the ovary to further decrease NPR2 activity. These results indicate that Sgpl1 deletion decreases NPR2 activity in the ovary by accumulating S1P. It has been reported that knocking out NPR2 causes the arrest of early follicle development8. Thus, deletion of Sgpl1 increases S1P levels, contributing to NPR2 inactivity that leads to follicle development failure.Disruption of the dynamic balance in cellular S1P leads to defects in reproductionSgpl1 deletion led to a reduction in PCNA-, BrdU-, and Ki-67-positive granulosa cells, suggesting the inhibition of granulosa cell proliferation. NPPC/NPR2 acts through the cGMP signaling pathway and can promote the growth of cultured preantral follicles in mice and rats31. Therefore, the inactivity of NPR2 by Sgpl1 deletion may reduce the proliferation of granulosa cells, resulting in the obstacle of early follicle growth. On the other hand, Sgpl1 deletion increased the mRNA and protein levels of p21 in granulosa cells possibly by S1P, since S1P can increase the transcription of p21 in human MCF-7 breast cancer cells24. The increase in p21 may participate in the arrest of granulosa cell proliferation. Furthermore, our RNA-seq data showed that deletion of Sgpl1 downregulated the expression of tubulin in granulosa cells, which may impair gap junction formation by disturbing the function of connexin-43 (ref. 28). The downregulation of transcripts related to cell metabolic enzymes  were decreased, and that the mRNA levels of Patz1 and Gli1 (ref. 35) were increased in Sgpl1 KO mice, indicating spermatogenesis disorder. We used testicular tissue to measure the S1P levels because of the limited number of Leydig cells in Sgpl1 KO mice. Anyway, the increase in S1P levels in the testicular microenvironment could act on Leydig cells. It is needed to further study S1P levels and SGPL1 functions in Leydig cells and germ cells, respectively.The reduction in Leydig cell populations and testosterone levels could result in germ cell apoptosisSgpl1 deletion increased S1P levels and then inhibited early follicular development and spermatogenesis mainly by NPR2 inactivity and p21 expression, respectively. Thus, SGPL1 plays a crucial role in mouse germ cell development by regulating S1P levels. Sgpl1 recessive mutations in humans cause primary gonadal failure12. Therefore, the mechanism of SGPL1 in mice provides a potential implication for the diagnosis and treatment of clinical infertility.In our study, Sgpl1 deletion mice of the C57/BL6 background were generated by using CRISPR/Cas9 gene editing technology  mice were purchased from the Laboratory Animal Center of the Institute of Genetics and Developmental Biology . ogy Fig. . In some7. Then, follicles were cultured in 96-well plates supplemented with S1P (10–30 μM) and/or FSH (25 ng/ml) for 4 days, and the diameters of follicles were measured daily. The follicle culture medium was bicarbonate-buffered minimum Eagle medium-alpha with Earle balanced salts  supplemented with 1% ITS (I3146), 100 UI/ml penicillin-streptomycin, 0.23 mM pyruvate, and 3 mg/ml bovine serum albumin. For the analysis of calcium levels and the binding affinity of NPR2 for NPPC, preantral follicles were cultured without or with S1P (30 μM) for 2 h. For the analysis of the gene, protein, S1P, and cGMP levels, the ovaries were collected from 12- to 14-day-old WT and KO mice. In some experiments, ovaries isolated from 10-day-old mice were cultured without or with S1P (30 μM) for 4 days. For the BrdU assay, the ovaries were cultured with 1 μM 5-bromo-2′-deoxyuridine for 3 h. The ovaries were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Thermo Fisher Scientific) supplemented with ITS and penicillin-streptomycin. All cultures were carried out at 37 °C in an atmosphere of 5% CO2.Secondary preantral follicles (100–110 μm) were stripped from 12-day-old mice36. Briefly, the internal tissue of testis was digested with collagenase II (C6885). The resuspended cells were filtered and layered onto a 8 ml solution of 60, 37, 26, and 21% Percoll (P4937), and centrifuged at 3000 × g for 30 min at 4 °C. The Leydig cells were harvested at the interface between 60 and 37% Percoll, and the purity was identified by HSD3B immunofluorescence staining. The Leydig cells were cultured in DMEM/F12 medium supplemented with S1P, JTE-013, and/or VPC23019  for the indicated time.Leydig cells were isolated from 21- to 23-day-old mice according to a previous report with slight modification7. The sections of testes were stained with hematoxylin and eosin.For immunofluorescence, the tissue samples were fixed in 4% paraformaldehyde (PFA) at 4 °C overnight, embedded in paraffin, and sectioned at 5 μm. The sections were dewaxed, rehydrated, and subjected to antigen retrieval. The Leydig cells were fixed with 4% PFA for 20 min and permeabilized with phosphate-buffered saline (PBS) solution containing 0.3% Triton X-100 (PBST) for 30 min. After blocking with 10% normal donkey serum, the sections or cells were incubated with primary antibodies Table  and thenTotal proteins were extracted in WIP buffer  with 1 mM phenylmethylsulfonyl fluoride on ice. 30 μg protein from each sample were separated by 10% SDS-polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes . After blocking with 5% nonfat milk, the membranes were incubated with primary antibodies Table  overnighTotal RNA was extracted and purified by the RNeasy micro-RNA Isolation Kit , and then reverse transcribed into cDNA by the QuantiTect Reverse Transcription System (Qiagen). qRT-PCR was conducted and analyzed on an ABI 7500 instrument  using a standard protocol. For single-cell RNA-seq analysis, RNA was extracted from granulosa cells of 14-day-old WT and KO mice, and transcriptome analysis was performed by Annoroad Gene Technology Co., Ltd. . For RNA-seq analysis, RNA was extracted from testes of 21-day-old WT and KO mice, and analyzed by Annoroad Gene Technology Co., Ltd. . The RNA-seq data were partially verified by qRT-PCR and western blotting. The qRT-PCR primers are listed in Supplementary Table Apoptotic cells were detected using the Click-iT Plus TUNEL Assay . Briefly, the rehydrated sections or cultured cells were fixed with 4% PFA, permeabilized, and then incubated with a TUNEL reaction mixture. After washing with PBS, the smaples were counterstained with DAPI. The incorporated fluorescence was visualized under the confocal microscope.5. The fluorescence was detected by the confocal microscope. All quantifications were performed using the Nikon NIS Elements BR 5.10 software. The fluorescence intensity represents the intracellular calcium levels (pseudocolor) and the binding affinity of NPR2 for NPPC (green), respectively.Preantral follicles were incubated in culture medium containing 5 µM Fluo 3-AM  for the detection of calcium levels, or incubated in culture medium containing 100 nM mono-5-(and 6)-carboxyfluorescein-labeled NPPC  for the detection of the binding affinity of NPR2 for NPPC as described previouslyOvarian tissues were solubilized in 200 µl of 5% trichloroacetic acid (TCA). After removing TCA, the samples were used for cGMP assay by a cGMP enzyme immunoassay kit obtained from Cayman Chemicals .37. In total, 10 µl of the supernatant was injected into the LC-MS/MS system  equipped with an electrospray ionization source and multiple reaction monitoring (MRM) for analysis. The concentrations on the S1P standard curve were 0.1, 0.5, 1, 5, and 10 ng/ml. LC separation was performed on a Kinetex C18 column   at 35 °C. The mobile phase consisted of 0.1% formic acid-water (eluent A) and acetonitrile (eluent B) at a flow rate of 0.3 ml/min. The retention times of S1P and C17-S1P were 9.07 and 8.85 min, respectively. MS analysis was performed in positive ion mode with the following settings: curtain gas, 35 psi; ion spray voltage, 4500 V; and ion source heater temperature, 550 °C. According to the MRM, the ion transitions of S1P were 380.2 → 264.1 and those of C17-S1P were 366.1 → 250.1, with the same DP of 71 V and CE of 19 V.The measurement of S1P was performed on liquid chromatography-tandem mass spectrometry (LC-MS/MS) with D-erythro-sphingosine-1-phosphate (S1P) as the standard and C17-D-erythro-sphingosine-1-phosphate  as the internal standard. The method for sample processing is consistent with the previous reportt test or one-way analysis of variance. Values of p < 0.05 were considered significant.All experiments were independently performed at least three times with different mice or cell preparations. The qualitative data reported are representative results obtained in replicate experiments and presented as the mean ± SEM. Statistical analysis was performed using SPSS 20.0 . The differences among groups were determined using Student’s Supplementary Figure legendsSupplementary TablesFigure S1Figure S2Figure S3Figure S4Figure S5Figure S6Figure S7Figure S8Figure S9Figure S10Figure S11


High-risk human papillomavirus (HPV) infection is the cause of almost all cervical cancers. HPV16 is one of the main risk subtypes. Although screening programs have greatly reduced the prevalence of cervical cancer in developed countries, current diagnostic tests cannot predict if mild lesions may progress into invasive lesions or not. In the current cross-sectional and longitudinal clinical study, we found that the HPV16 E7-specific T cell response in peripheral blood mononuclear cells of HPV16-infected patients is related to HPV16 clearance. It contributes to protecting the squamous interaepithelial lesion (SIL) from further malignant development. Of the HPV16 infected women enrolled (n = 131), 42 had neither intraepithelial lesion nor malignancy (NILM), 33 had low-grade SIL, 39 had high-grade SIL, and 17 had cervical cancer. Only one of 17 (5.9%) cancer patients had a positive HPV16 E7-specific T cell response, dramatically lower than the groups of precancer patients. After one year of follow-up, most women  with persistent HPV infection did not exhibit a HPV16 E7-specific T cell response. Furthermore, 3 malignantly progressed women, one progressed to high-grade SIL and two progressed to low-grade SIL, were negative to the HPV16 E7-specific T cell response. None of the patients with a positive HPV16 E7-specific T cell response progressed to further deterioration. Our observation suggests that HPV16 E7-specific T cell immunity is significant in viral clearance and contributes in protection against progression to malignancy. Cervical cancer is a malignant tumor that seriously threatens the life and health of women. Globally, approximately 500,000 women are diagnosed with cervical cancer every year and of these 280,000 women die from cervical cancer . It is aHPV-related carcinogenesis is not a uniform process. The “integration” of the virus into the host cell increases gene instability, marking the beginning of malignant transformation . It takeAmong all high-risk HPVs, more than half of cervical cancer patients around the world are infected with HPV16 . The resIn this study, we used the recombinant overlapping peptide protein of HPV16 E7 (ROP-HPV16 E7) and the E7 overlapping polypeptides as stimulants in the Enzyme-linked immunospot (ELISPOT) assay to evaluate the HPV16E7 specific-T lymphocyte response in peripheral blood mononuclear cells (PBMCs) of patients. The results showed that the low HPV16 specific T cell response in peripheral blood was significantly correlated with persistent viral infection. Our study suggests that the HPV16-specific T cell response in peripheral blood can effectively predict clinical outcome.E. coli, is able to replace the costly, and difficult to manufacture, quality-controlled pool of HPV16 E7 peptides, in the interferon-γ release assay (IGRA).In our previous study, ROP-HPV16E7 effectively stimulated the HPV16 E7-specific T cell response in mice immunized with the HPV protein . In thisA total of 131 female patients with HPV16 (type 16) infection was recruited for the study. At the time of enrollment, the IGRA-based HPV16 E7-specific T cell response of PBMCs was assessed and cervical biopsy diagnosis was performed. There were 42 patients without intraepithelial lesions or malignant lesions (NILM), 33 patients with low-grade SIL (LSIL), 39 patients with high-grade SIL (HSIL), and 17 patients with cervical cancer Table 1.We first performed an HPV type16 E7 ROP-based IGRA to assess the status of HPV-related T cell immunity. In the NILM group, 11 patients (26.2%) tested positive for HPV16 E7-specific T cell response and 31 patients (73.8%) had a negative response. In the LSIL group, 14 patients (42.4%) tested positive and 19 patients (57.6%) tested negative for the HPV16-specific T cell response. In the HSIL group, 15 patients (38.5%) were positive and 24 (61.5%) negative for the HPV16 E7 T cell response. All HSIL patients underwent surgery to remove the local pathological lesions. Of the 17 cervical cancer patients, only 1 (5.9%) patient showed a positive HPV16 E7-specific T cell response, and 16 (94.1%) patients presented a negative response Table 2.To determine whether the HPV or HPV peptide-specific T cell response was relevant to the clearance of the persistent HPV infection in different pathogenetic groups, at the end of the one-year follow-up period, HPV genotyping was performed on cervical exfoliated cell samples. In the NILM group, only 2 of 11 patients (18.2%) with a positive HPV16 E7-specific T cell response at the start of the study sustained HPV16 infection. However, 11 of 31 patients (35.5%) who were negative for the HPV16 ROP-specific T cell response harbored a persistent infection. The persistent infection rate was 1.95 times higher than that of patients with positive responses (P = 0.005). In the LSIL group, 2 of 14 patients (14.3%) with a positive HPV16 ROP-specific T cell response sustained a HPV16 infection, while 12 of 19 patients (63.2%) with a negative HPV16-specific T cell response were persistently infected. The persistent infection rate was 4.42 times higher than that of patients with positive response (P <0.0001). In the HSIL group, 39 patients with HPV16 virus infection and high lesions were diagnosed at the time of enrollment, and all these patients underwent surgery. After one year, only 1 of 15 patients (6.7%) with a positive HPV16 specific T cell response sustained HPV16 infection, but 8 of 24 patients (33.3%) with a negative T cell response sustained HPV16 infection. The persistent infection rate was 4.97 times higher than in patients with positive HPV16-specific T cell response (P <0.0001) Table 3.To study the influence of the T-cell immunity response on HPV pathogenesis, at the end of this study, a cervical biopsy was performed. The results showed that in all three groups , none of the patients with positive specific T cell response against HPV16 E7 presented any further disease deterioration Table 4.A total of 114 women completed the 12-month study. At the beginning of their hospital visit, all were positive for HPV16 infection by PCR test. Forty patients (35.1%) were positive for the HPV16 E7 ROP-specific T cell response. The remaining 74 patients (64.9%) were negative for the T cell response Table 5.+ T cells, CD4+ T cells, CD4+:CD8+ ratio, CD8+ T cells expressing PD-1 and regulatory T cells expressing Foxp3. The results showed no correlation with prognosis . All images used in this study were acquired using an Olympus BX45 microscope equipped with 20× objective . All histological sections were reviewed by a single histopathologist for the purpose of this study, who was blinded to other clinical information. The cases were classified as NILM, LSIL, HSIL, or cancer.Four synthetic overlapping peptides covering the entire sequence of the HPV16 E7 protein were synthesized : Peptide 1, I R T L E D L L M G T L G I V C P I C S Q K P; peptide 2, M H G D T P T L H E Y M L D L Q P E T T D L Y C Y E Q L N D S S E E E; peptide 3, E Q L N D S S E E E D E I D G P A G Q A E P D R A H Y N I V T F C C K; and peptide 4, H Y N I V T F C C K C D S T L R L C V Q S T H V D I R T L E D L L M G.The ROP-HPV16 E7 recombinant overlapping peptide is an artificial protein that contains E7 overlapping peptides linked to LRMK (a cathepsin S cleavage site). ROP-HPV16 E7 were expressed and purified as described elsewhere . Brieflyg for 25 min. The PBMC layer was transferred to a new tube and was washed twice with RPMI 1640 medium.Heparin-treated human blood (10 ml) from patients was carefully added to the lymphocyte-separation medium (density = 1.077) and centrifuged at 1,500×5 cells/well) were stimulated overnight with 2 µM ROP-HPV16 E7 or with mixed E7 peptides in anti-IFN-γ-Ab precoated plates . Cells were discarded and biotinylated anti-IFNγ antibodies were added for 2 h at room temperature, followed by an additional 1 h incubation at room temperature with enzyme-labeled strepavidin. After the color developed, the reaction was stopped by washing the plates with tap water, and the plates were air dried. Spots were counted with an Elispot reader .The assays were performed using ELISPOT kits . PBMCs , FITC-conjugated anti-human CD8 , PE-conjugated anti-human CD279 monoclonal antibody  and FVS620  were added and incubated in the dark for 15 min at room temperature. For FoxP3 staining, PBMCs were first stained with FITC-conjugated anti-Human CD4 monoclonal antibody , APC-conjugated anti-Human CD25 monoclonal antibody  and FVS620  for 15 min in the dark at room temperature. After fixation and permeabilization, the cells were stained with PE-conjugated anti-human FoxP3 monoclonal antibody  overnight at 4°C. The cells were then washed three times with 1 ml of FACS buffer, resuspended in 0.5 ml of FACS fixing buffer  and acquired using CytoFLEX S flow cytometer . The data was analyzed by FlowJo software version VX .PBMCs were washed once with 1 ml ice-cold FACS buffer (2% FCS in PBS) and the cell density was adjusted to 1 × 10t-test for continuous variables and Chi-square test for categorical variables. A P-value <0.05 in the two-sided test was considered statistically significant. Statistical analyses were performed using IBM SPSS Statistics version 25 . We used the The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Ethics Committee of Changzhou Maternal and Child Health Hospital. The patients/participants provided their written informed consent to participate in this study.SJ and WL designed and wrote the manuscript. LZ collected patient samples and performed most of the experiments. XinS and WL carried out ELISPOT analysis. QZ performed flow cytometry assays. ZM and XiaS helped to collect patient samples. ZM and AJ participated in the statistical analysis; RZ and JZ participated in the experimental design and project management. All authors contributed to the article and approved the submitted version.This research was supported by grants from the general scientific research project of Jiangsu Provincial Health Commission (H2018018).Authors XinS, QZ, AJ and RZ were employed by company Oxford Vacmedix Co. Ltd. Author WL was employed by company Oxford Vacmedix Co. Ltd. and Shanghai JW Inflinhix Co. Ltd.The remaining 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


However, it has been reported recently that H2S, together with nitric oxide and carbon monoxide, is an important gas signal transduction molecule. H2S has been reported to play a protective role in many diabetes-related diseases, but the mechanism is not fully clear. Recent studies indicate that H2S plays an important role by regulating autophagy in many diseases including cancer, tissue fibrosis diseases and glycometabolic diseases; however, the related mechanism has not been fully studied. In this review, we summarize recent research on the role of H2S in regulating autophagy in diabetic-related diseases to provide references for future related research.Autophagy is a vital cell mechanism which plays an important role in many physiological processes including clearing long-lived, accumulated and misfolded proteins, removing damaged organelles and regulating growth and aging. Autophagy also participates in a variety of biological functions, such as development, cell differentiation, resistance to pathogens and nutritional hunger. Recently, autophagy has been reported to be involved in diabetes, but the mechanism is not fully understood. Hydrogen sulfide (H Accordiganelles ,13. Autoganelles . Howeverganelles ,16. Autoganelles ,18,19, cganelles , neurodeganelles , infectiganelles . HoweverDiabetes is an important metabolic disease. Its prevalence rate is significantly higher than before. In the 1990s, the number of diabetic patients worldwide was 135 million, and it may rise to 300 million by 2025 . Diabete2S) has been understood as a colorless gas with a smell of rotten eggs, which is recognized as a toxic gas and environmental pollutant. Recently, along with carbon monoxide (CO) and nitric oxide (NO), H2S is considered as the third gasotransmitter [2S is produced from L-cysteine and/or L-homocysteine catalyzed by cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS), cysteine aminotransferase and 3-mercaptopyruvate sulfurtransferase (3-MST) [2S [2S has been reported to have many biological functions including antiapoptosis [2S is particularly important.For decades, hydrogen sulfide (Hnsmitter . Endogen (3-MST) . CystathMST) [2S  Figure S has beepoptosis , antioxipoptosis , relaxinpoptosis ,31 and apoptosis . The excpoptosis . Therefo2S improves diabetes-accelerated atherosclerosis through inhibiting oxidative stress via Kelch-like ECH-associated protein 1(Keap1) sulfhydrylation of Cys151 to activate nuclear factor erythroid-2 related factor 2(Nrf2) signaling [2S also alleviates diabetes-induced atrial remodeling and atrial fibrillation by activating the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt)/eNOS pathway [2S affecting diabetes is the effect of H2S on the pancreas. H2S can be produced in the pancreas. In pancreatic beta cells, H2S inhibits high glucose (HG)-induced insulin release and decreases HG-induced apoptosis of pancreatic islets. H2S also protects pancreatic beta cells against glucotoxicity by increasing glutathione content and reducing ROS production. However, the high concentrations of H2S can promote the apoptosis of pancreatic beta cells [2S deficiency has been reported to protect the pancreas β cells from apoptosis and delay the development of streptozotocin (STZ)-induced type 1 diabetes mellitus [2S on different types of diabetes may be different.It has been reported that exogenous Hignaling . H2S als pathway . At presta cells . Endogenmellitus . HG is o2S plays an important role by regulating autophagy in many diseases including cancer, tissue fibrosis diseases and glycometabolic diseases [2S regulating autophagy in diabetic-related diseases to provide reference for future related research.Autophagy is also involved in diabetes. Liraglutide is an acylated glucagon-like peptide-1 analogue, which has a 97% amino acid homology with natural glucagon-like peptide-1 and has been widely used in the treatment of type 2 diabetes mellitus . Liraglu2S could improve DCM through ameliorating diastolic function and increasing H2S production. Similar results were obtained in vivo. Mechanism research showed that exogenous H2S inhibited oxidative stress by decreasing ROS production and upregulating the expression levels of mitochondrial catalase (Mito-CAT) and manganese-dependent superoxide dismutase (Mn-SOD). The results of the experiment of the antioxidation mechanism showed that exogenous H2S had no significant effect on Nrf2 nuclear translocation, meaning it could be excluded that the antioxidant effect of exogenous H2S was mediated by the keap-1/Nrf2 signaling pathway. Exogenous H2S also increased autophagy by upregulating the expression level of Beclin1, microtubule associated protein 1 light chain 3 II (LC3II) and autophagy associated protein 7 (Atg7), promoting the degradation of autophagosome content and decreasing the expression level of p62. In order to explore a new explanation for the H2S antioxidant effect, the Keap-1 effect on promoting ubiquitin aggregate clearance was studied. The results showed that exogenous H2S decreased the ubiquitylation levels of Keap-1, CAT and SOD, which might be the reason why exogenous H2S had the antioxidant effect, while Keap-1 siRNA inhibited the effects of exogenous H2S on autophagy in the cardiomyocytes of diabetic rats. 3-MA (an autophagy inhibitor) abolished the antioxidant effect of exogenous H2S, suggesting that exogenous H2S promoted ubiquitin aggregation clearance by upregulating autophagy through activating keap-1. 1,4-Dithiothreitol, a reducing agent of disulfides, reduced the expression level of keap-1 by promoting its ubiquitination level and counteracted the effects of exogenous H2S on keap-1, ubiquitin aggregate clearance and oxidative stress in HG-induced H9C2 cells. Moreover, exogenous H2S could promote the formation of disulfide between two keap-1 molecules, suggesting that exogenous H2S suppressed Keap-1 ubiquitylation through promoting its disulfide formation. From the above results, it can be deduced that exogenous H2S ameliorates DCM by promoting ubiquitin aggregation clearance through promoting autophagy via ubiquitylation of Keap-1, which provides a new mechanism for the antioxidative stress of H2S [2S improves DCM through autophagy is worth studying. Moreover, more research is needed on how exogenous H2S inhibits keap-1 ubiquitination to promote autophagy.Diabetes affects the heart through a variety of mechanisms including metabolic disorders, abnormal subcellular components, microvascular damage and dysfunction of cardiac autonomic nerve damage ,41,42. Es of H2S . In the 2S inhibited NLRP3 inflammasome-mediated inflammation by activating autophagy via the AMPK/mTOR signaling pathway [2S also can improve DCM by regulating autophagy through the AMPK/mTOR signaling pathway. Studies of Fan Yang and colleagues have shown that exogenous H2S could ameliorate left ventricular systolic dysfunction, increase the cell survival rate and inhibit cardiomyocyte apoptosis and oxidative stress to improve DCM. Meanwhile, exogenous H2S promoted autophagy by increasing autophagic vesicles and the expression level of autophagic-related proteins. Mechanism studies showed that exogenous H2S activated the AMPK/mTOR signaling pathway through increasing the p-AMPK/AMPK ratio and decreasing the p-mTOR/mTOR ratio in a diabetes model. Compound C, an inhibitor of AMPK, suppressed the activity of mTOR, suggesting that mTOR is downstream of AMPK in the regulation of autophagy by exogenous H2S. Compound C and AMPK-siRNA both inhibited H2S-promoted autophagy in HG-induced H9C2 cells, increased apoptosis and aggravated cell injury, indicating that exogenous H2S alleviated DCM by activating autophagy through activating the AMPK/mTOR signaling pathway [2S-induced autophagy and the AMPK/mTOR signaling pathway needs further study in diabetes.Adenosine 5′-monophosphate activated protein kinase (AMPK) is a serine/threonine kinase and regulates many physiological and pathological processes including apoptosis, proliferation, cell growth, migration and differentiation ,56. The  pathway . Similar pathway . The rel2S reversed the diabetes-induced changes, suggesting that exogenous H2S could improve myocardial fibrosis induced by diabetes. Mechanism studies revealed that exogenous H2S inhibited diabetes-induced autophagy through decreasing the number of autophagosomes and the expression levels of Beclin-1, Atg3, Atg5 and Atg16. The expression of PI3K and AKT1 was inhibited by diabetes, while exogenous H2S reversed the changes, suggesting exogenous H2S activated the PI3K/AKT1 signaling pathway suppressed by diabetes [2S mitigates diabetes-induced myocardial fibrosis by inhibiting autophagy via activating the PI3K/AKT1 signaling pathway, which needs to be further studied by using inhibitors to suppress autophagy and the signaling pathway.Myocardial fibrosis is caused by the excessive deposition of collagen in the extracellular matrix (ECM). It is one of the main pathological features of DCM, which can lead to diastolic and systolic dysfunction in patients with DCM ,61. Diabdiabetes . It has diabetes . Therefo2S can protect the myocardium by inhibiting overactivated autophagy in the HG environment. It has been reported that myocardial cell aging is closely related to myocardial fibrosis [2S could reverse the above changes, indicating that exogenous H2S could improve diabetic myocardial fibrosis, inhibit myocardial cell senescence and promote autophagy in diabetes models [2S alleviates diabetic myocardial fibrosis through suppressing myocardial cell senescence via activating autophagy. Sirtuin6 (SIRT6), a member of the NAD+-dependent deacetylase family, has been reported to play an important role in senility [2S activated the SIRT6/AMPK signaling pathway suppressed by HG through upregulating SIRT6 and AMPK protein expression, while the SIRT6 inhibitor or AMPK inhibitor reversed the effects of exogenous H2S on autophagy and senescence, suggesting that exogenous H2S could improve diabetic fibrosis by suppressing myocardial cell senescence via activating autophagy through activating the SIRT6/AMPK signaling pathway [Exogenous Hfibrosis ,65. Yalis models . In addis models . From thsenility . AMPK ansenility . Mechani pathway . The ref2S mitigated dysfunction of arterial endothelial cells by decreasing the expression levels of marker proteins including Von Willebrand factor (vWF), Integrin beta-1 (ITGβ1) and GP1β A and reduced apoptosis of rat aortic endothelial cells (RAECs) by decreasing the expression levels of apoptosis-related proteins and mitigating mitochondrial damage in a diabetes model. Meanwhile, N-acetylcysteine (NAC), a scavenger of ROS, had similar effects to exogenous H2S, indicating that ROS mediated the above effects of exogenous H2S. The LC3II/I ratio is an indicator of autophagy formation and maturity; Atg7 mediates the formation of LC3-II; Lamp2 is a lysosomal membrane protein, which can be used to monitor the fusion of autophagosomes and lysosomes. HG could increase the LC3II/I ratio and the expression levels of Atg7 and Lamp2 and decrease the expression level of P62 in HG-induced RAECs, while exogenous H2S reversed the changes. Moreover, the inhibition of autophagy with 3-MA and Atg7 siRNA reduced RAEC apoptosis induced by HG. From the above, it can be inferred that exogenous H2S improve HG-induced ECD through inhibiting autophagy induced by HG via reducing ROS production. Mechanism research showed that HG increased the ratio of p-AMPK/AMPK, while exogenous H2S reversed the change. Compound C also suppressed HG-induced autophagy, suggesting that exogenous H2S inhibited autophagy by suppressing the HG-induced AMPK signaling pathway [2S ameliorated mitochondrial damage treated with HG by increasing ATP production, respiratory complex activity and the expressions and activities of SOD and CAT, suggesting that exogenous H2S inhibited HG-induced AMPK activation through improving mitochondrial damage [2S promoted Nrf2 nuclear transfer [2S improve ECD through inhibiting autophagy via the Nrf2-ROS-AMPK signaling pathway, which provides a new way to treat diabetes-induced ECD.Endothelial cell dysfunction (ECD) can induce the impairment of endothelial cell barrier function and vasodilation and plays a vital role in the complications of diabetes ,71. It h pathway . One stu pathway , while el damage . Nrf2 isl damage ,78. The transfer . In conc2S ameliorated renal fibrosis by decreasing the expression levels of MMPs, collagen IV and TIMP in diabetic kidney tissue. Exogenous H2S also promoted autophagy by upregulating the expression levels of autophagy biomarkers, including LC3, Atg3, Atg5, Atg7, Atg12 and Atg16 [2S on diabetes-induced renal fibrosis. Different from the above, another study showed downregulation of autophagy in diabetes-induced renal fibrosis [2S reduced the expression levels of TGFβ1, nuclear factor of kappa B (NF-κB) and AKT in diabetic kidney tissue, suggesting the TGFβ1, NF-κB and AKT pathways may mediate the effects of exogenous H2S on autophagy in improving diabetes-induced renal fibrosis [Diabetic nephropathy (DN) is the main cause of end-stage renal disease ,80. It ind Atg16 . Deregulnd Atg16 . Therefofibrosis . This difibrosis , which n2S in diabetic patients and diabetic rats were decreased. ALA treatment could increase the level of plasma H2S in diabetic rats. ALA could also reverse the inhibitory effect of propargylglycine  on endogenous H2S production; furthermore, the expression level of CSE was decreased in diabetic rats, indicating that ALA could promote endogenous H2S production. ALA could protect VSMCs, while PPG reversed the ALA protective effect, suggesting that ALA protected VSM in diabetic rats by promoting endogenous H2S production. The expression levels of LC3BII/LC3BI, Beclin-1 and phosphorylated AMPK were increased, and the expression levels of p62 and phosphorylated mTOR were decreased in diabetic rats, while ALA could reverse the changes. Moreover, PPG attenuates ALA-induced inhibition of autophagy and the AMPK/mTOR pathway. Therefore, it can be inferred that ALA suppresses autophagy by suppressing the AMPK/mTOR pathway of VSM in diabetic rats through increasing the endogenous H2S level. Similar to in vivo experiments, exogenous H2S and ALA significantly inhibited autophagy and increased cell viability in HG-induced VSMCs. Rapamycin (an autophagy activator) and AICAR (an AMPK activator) both abolished the effects of exogenous H2S and ALA on autophagy and cell viability. In addition, AICAR activated AMPK and decreased the level of phosphorylated mTOR protein, and compound C further enhanced the above effects of exogenous H2S and ALA. Collectively, it can be deduced that exogenous H2S and ALA improve diabetic dysfunctional VSM by inhibiting autophagy via the AMPK/mTOR pathway, which offers a new strategy for the treatment of diabetic macroangiopathy [Diabetic macroangiopathy can lead to cerebrovascular disease, which is one of the main causes of death in diabetic patients. Dysfunctional vascular smooth muscle (VSM) plays an important role in diabetic macroangiopathy ,87. The giopathy .2S has antidepressant effects in diabetic rats [2S can improve diabetic depression by regulating autophagy, Hai Yao Liu and colleagues committed to a series of experiments, and the results showed that exogenous H2S increased the expression levels of BDNF and p-TrkB proteins in the hippocampus of diabetic rats. K252a (an inhibitor of the BDNF-TrkB pathway) abolished the antidepressant effects of H2S. Furthermore, K252a inhibited exogenous H2S-promoted hippocampal autophagy in diabetic rats by decreasing the number of autolysosomes and Beclin-1 expression and increasing P62 expression in the hippocampus of diabetic rats. In conclusion, exogenous H2S can improve diabetic depression by promoting autophagy via the BDNF-TrkB pathway [2S can prevent diabetes depression by improving damaged hippocampal neurons and reducing synaptic plasticity-related proteins.Many studies have reported a high prevalence of depression in diabetic patients ,94, and tic rats . Brain-dtic rats , and platic rats . Moreovetic rats . To prov pathway , which p2S in regulating autophagy in diabetic-related diseases as follows: (1) exogenous H2S ameliorates DCM by promoting ubiquitin aggregation clearance through promoting autophagy via ubiquitylation of Keap-1; (2) exogenous H2S alleviates DCM by activating autophagy through activating the AMPK/mTOR signaling pathway; (3) exogenous H2S mitigates diabetes-induced myocardial fibrosis by inhibiting autophagy via activating the PI3K/AKT1 signaling pathway; (4) exogenous H2S could improve diabetic-induced myocardial fibrosis by suppressing myocardial cell senescence via activating autophagy through activating the SIRT6/AMPK signaling pathway; (5) exogenous H2S can improve HG-induced ECD through inhibiting autophagy via the Nrf2/ROS/AMPK signaling pathway; (6) exogenous H2S improves diabetes-induced renal fibrosis by activating autophagy via inhibiting the TGFβ1/NF-κB/AKT pathways; (7) exogenous H2S improves diabetic dysfunctional VSM by inhibiting autophagy via suppressing the AMPK/mTOR pathway; (8) exogenous H2S can improve diabetic depression by promoting autophagy via activating the BDNF/TrkB pathway (2S sometimes activates autophagy and sometimes inhibits autophagy. The reason may be related to the severity of diabetes. In the early stage of diabetes, when the disease is mild, H2S often activates autophagy to protect cells, and with the progress of the disease, H2S inhibits the overactivated autophagy to protect cells. In addition, under physiological conditions, the level of autophagy in different tissues is different, which may be another reason for the abovementioned findings. The effect of autophagy on cells is a “double-edged sword” because if autophagy is maintained at a high level, autophagy will lead to autophagy death. Therefore, the role of autophagy in different complications of diabetes needs further study.In this review, we summarized the role of exogenous H pathway . From th2S can inhibit the inflammation of liver cells mediated by the NLRP3 inflammasome by upregulating autophagy [2S can regulate autophagy through the NLRP3 inflammasome in diabetic-related diseases is a very worthy topic to study. With the further research on the improvement of diabetes by H2S regulating autophagy, it may provide a new strategy for the treatment of diabetes.Our previous studies have shown that exogenous Hutophagy . Moreoveutophagy . Therefo


H3F3A K27M mutation using digital droplet PCR. In 10 patients, CSF was obtained by lumbar puncture at presentation. A definitive detection of H3F3A K27M mutation was achieved in only one case (10%); H3F3A K27M mutation was suspected in three other cases (30%). H3F3A K27M mutation was detected in two patients in CSF obtained by ventricular tap during a ventriculo-peritoneal shunt for obstructive hydrocephalus. Cases in which a definitive assessment was possible (definite H3F3A K27M or definite H3F3A wildtype) tended to be younger  and have a higher concentration of CSF protein  compared to nondefinite cases. Low proliferation and apoptotic rates seemed to be characteristics of DMG unfavorable for liquid biopsy. More advanced lesions with necrosis and evidence of dissemination were unlikely to be candidates for lumbar puncture due to the fear of exacerbating obstructive hydrocephalus. Methods to safely sample CSF and a more sensitive detection of ctDNA are necessary for reliable liquid biopsy of DMG at presentation.Recent studies have suggested the feasibility of detecting H3K27M mutations in the cerebrospinal fluid of diffuse midline glioma (DMG) patients. However, cerebrospinal fluid from patients in these studies were collected mainly during biopsy, ventriculo-peritoneal shunt procedures or postmortem. We assessed circulating tumor DNA (ctDNA) extracted from cerebrospinal fluid (CSF) and plasma in a series of 12 radiographically suspected and/or pathologically confirmed diffuse midline glioma patients and assessed for  HIST1H3B (H3.1K27M) or H3F3A (H3.3K27M) have been reported in 50 to 80% of diffuse intrinsic pontine gliomas and thalamic gliomas [Recurrent H3B H3.1K7M or H3F gliomas ,5,6; thuMYD88 mutation status in circulating tumor DNA (ctDNA) extracted from cerebrospinal fluid (CSF) and biopsied tissue in 21 primary and secondary central nervous system lymphoma patients using droplet digital PCR (ddPCR) [We have previously reported a 100% match of the  (ddPCR) ,10 or ne (ddPCR)  of ctDNAg for 10 minutes to remove cells, and the supernatant was stored at −80 °C within 30 minutes of collection. ctDNA was extracted using the Maxwell RSC ccfDNA Plasma Kit , according to the manufacturer’s instructions. For all samples, DNA was dissolved in 60 μL of elution buffer and stored at −20 °C until further use. ddPCR reagents and primer/probe mix for H3F3A K27M were purchased from Bio-Rad . A 20 μL PCR mix, composed of 10 μL 2× ddPCR Supermix for Probes , 1 μL ddPCR Mutation Assay (Bio-Rad) and 9 μL ctDNA, was loaded into sample wells of an eight-channel disposable droplet generator cartridge (Bio-Rad). An additional 70 μL of droplet generation oil (Bio-Rad) was loaded into the oil well for each channel. After droplet generation, the droplets were transferred into a 96-well PCR plate and subjected to thermal cycling. Amplification was carried out on the 20 μL reaction mixture on the QX-200 ddPCR system (Bio-Rad). After PCR, the 96-well PCR plate was subjected to the QX-200 droplet reader (Bio-Rad), and data were analyzed by QuantaSoft analysis software (Bio-Rad). H3F3A K27M-mutation specific signals were generated in the hexachloro-fluorescein channel. We considered definite mutant cases to have a fractional abundance of 0.1% or more and to have three or more mutant droplets and/or wildtype droplets detected. Cases in which only one or two mutant droplets were detected were considered H3K27M-mutant-suspect. Cases where two or fewer wildtype droplets were detected were considered unreliable. ddPCR was performed in at least two separate wells for each case whenever possible. Surgical removal or biopsy was performed in two cases (cases #7 and #11) with genetic confirmation of H3F3A K27M.Twelve patients with radiographically suspected diffuse midline glioma, treated at the Department of Neurosurgery, Niigata University Hospital between November 2017 and January 2021, who underwent liquid biopsy, were enrolled in the study. Written consent was obtained from patients and families for liquid biopsy after approval from the institutional review board #G2018-0008). Detailed methods of ddPCR have been previously published [-0008. DeU test and tests for associations between different parameters by the Fisher’s exact test for 2 × 2 contingency tables. Statistical analyses were performed using GraphPad Prism 9 software . p < 0.05 was considered significant.Comparisons between two groups were carried out using the Mann–Whitney Lesions were located at the pons in eight patients, medulla oblongata in one patient, thalamus in two patients and temporal lobe in one patient . A liquiH3F3A K27M mutation was obtained in only one (10%) patient showing diffuse hyperintensity of the pons on T2-weighted MR images (H3F3A K27M was suspected in three (30%) other patients at presentation, but only one or two mutant droplets were detected from each well in these patients  cases, the ddPCR results were considered unreliable, with a sufficient number of mutant and wildtype droplets not being detected.Lumbar puncture was performed in 10 patients at presentation . HoweverR images A and an R images B. A highR images C. Exophypatients . Two casH3F3A K27M was obtained at recurrence from two patients, both in CSF obtained from ventricular tap during ventriculo-peritoneal shunting for obstructive hydrocephalus in a recurrent DMG case with heterogeneously enhancing residual lesion of the right thalamus  versus nondefinite (defined as H3F3A K27M-suspect or unreliable) cases  and to have a higher concentration of CSF protein .We compared the clinical, radiographical and CSF characteristics of cases in which there was a definite diagnosis (defined as either a definite e) cases . Due to HIST1H3B (H3.1K27M) or H3F3A (H3.3K27M) mutations have been reported in 50 to 80% of diffuse intrinsic pontine gliomas and thalamic gliomas [Recurrent H3B H3.1K7M or H3F gliomas ,5,6. ThuHIST1H3B K27M mutations in four out of six (66.7%) diffuse midline gliomas. In that study, CSF was either obtained from the ventricles by external ventricular drainage at tumor biopsy (n = 2), at reservoir placement (n = 2) or by reservoir tap during treatment (n = 2). Thus, CSF was not collected by lumbar tap before treatment in any of the cases. Subsequently, two large studies were reported. Pan et al. reported the detection of H3F3A K27M mutations (n = 27) or HIST1H3B K27M mutations (n = 5) in 32 out of 57 (56%) DMG patients by next generation sequencing (NGS). CSF was obtained from the cisterns during biopsy in 52 (91.2%) cases, from during ventriculo-peritoneal shunt procedures in two (3.5%) cases, and by lumbar puncture in only three (5.7%) cases [Three previous reports highlight the feasibility of detecting H3K27M mutations in DMG patients by ctDNA analysis in CSF and plasma ,10,11. H2 vs. 373 mm2) [R technology [An important study by Miller et al. detected ctDNA in CSF obtained by lumbar puncture in 42 out of 85 (49.5%) patients harboring WHO grade 2, 3 and 4 gliomas using NGS. ctDNA was detected from a higher percentage of grade 4 tumors (64% to 44%), in patients with a positive CSF cytology (18% vs. 0%), higher CSF protein concentration (72 vs. 56), higher number of prior resections (two vs. one), in recurrent cases (55% vs. 19%) and in larger-volume tumors (1553 mm373 mm2) . Additiochnology .MYD88 L265P mutations in CSF in primary and secondary CNS lymphoma patients using ddPCR, with a 100% match of the MYD88 L265P mutation status between tumor and CSF in 21 cases with available tumor samples [MYD88 L265P mutations from the plasma and urine of PCNSL patients [We have previously reported a high detection rate of patients . Since cpatients , reflectpatients , DMGs arpatients ,18, freqpatients .HIST1H3B K27M mutations [MYD88 L265P and a 100% match of the MYD88 mutation status between CSF and tumor. Therefore, we can safely say that, at present, detecting H3F3A K27M in CSF obtained by lumbar puncture in DMG patients is difficult. Using the Maxwell RSC ccfDNA Plasma Kit, up to 4 mL of ctDNA can be extracted in CSF instead of the 1 mL used in the present study, potentially improving the detection of small amounts of ctDNA. However, obtaining large amounts of CSF can be challenging and possibly harmful in these patients. The development of more sensitive methods, including next generation sequencing or more sensitive PCR methods, is necessary for the reliable detection of H3F3A K27M in DMG patients before the commencement of treatment.The limitations of the present study include the small sample size, low number of pathologically confirmed cases and inability to detect H3F3A K27M by ddPCR in CSF obtained by lumbar puncture in DMG patients at presentation is low. Diagnosis by liquid biopsy at presentation may be possible in younger patients with a high protein concentration in CSF or evidence of leptomeningeal disease. Liquid biopsy is an exciting alternative to biopsy in DMGs, but both case selection and the development of more sensitive methods are vital.In the present report, we found that the detection rate of


Nuclear transfer embryonic stem cells (ntESCs) hold enormous promise for individual-specific regenerative medicine. However, the chromatin states of ntESCs remain poorly characterized. In this study, we employed ATAC-seq and Hi-C techniques to explore the chromatin accessibility and three-dimensional (3D) genome organization of ntESCs. The results show that the chromatin accessibility and genome structures of somatic cells are re-arranged to ESC-like states overall in ntESCs, including compartments, topologically associating domains (TADs) and chromatin loops. However, compared to fertilized ESCs (fESCs), ntESCs show some abnormal openness and structures that have not been reprogrammed completely, which impair the differentiation potential of ntESCs. The histone modification H3K9me3 may be involved in abnormal structures in ntESCs, including incorrect compartment switches and incomplete TAD rebuilding. Moreover, ntESCs and iPSCs show high similarity in 3D genome structures, while a few differences are detected due to different somatic cell origins and reprogramming mechanisms. Through systematic analyses, our study provides a global view of chromatin accessibility and 3D genome organization in ntESCs, which can further facilitate the understanding of the similarities and differences between ntESCs and fESCs. Nuclear transfer embryonic stem cells (ntESCs) provide a resource of patient-matched cells for personalized therapies and disease models with less teratogenic, which hold enormous promise for regenerative medicine , 2. BothRecently, we and another group showed that abnormal 3D genome structures were present in SCNT embryos, including aberrant enhancer-promoter interactions of ZGA genes, stronger TAD boundaries and others , 19. Whe2+-free CZB medium supplemented with 10 mM SrCl2  and 5 μg/ml cytochalasin B  for 6 h. Cloned and fertilized embryos were cultured in G1-plus medium  at 37 °C in an atmosphere of 5% CO2 in air.Mice were housed in a temperature-controlled environment under a 12 h light: 12 h dark cycle. All animal procedures complied with the Animal Care and Use Committee of Huazhong Agriculture University (HZAUMO-2016-031). Fertilized embryos were obtained from C57BL/6 females mated with DBA/2. For SCNT, metaphase II (MII) oocytes were collected from 8–10 weeks old female B6D2F1 (C57BL/6×DBA/2) mice. SCNT was performed as previously described . BrieflyDerivation of ES cells was performed as previously described with a slight modification . BrieflyAll other experimental procedures were described in https://github.com/v587dexinxin/Nuclear-Transfer-scripts.Hi-C and ATAC-seq experiments were repeated at least two times and RNA-seq experiments were repeated at least three times. The raw data were processed mainly by using publicly available bioinformatics tools Table . And theThe detailed computational procedures were described in Supplemental Materials.Firstly, we derived ESCs from mouse fertilized and SCNT blastocysts as previously  Fig. 1A1A. In toJunb, Runx1/2 and Fra1/2) are enriched in C2 while the pluripotent TF motifs  are relatively enriched in C3  Table A, B. The C3 Fig. . Moreoventiation –26  are detected by both 4 C and cHi-C in ESCs, which are highly co-localized with loop 6 in ntESCs and fESCs  anchored around gene and CH12 )  show normal ESC morphologies and pluripotent gene expressions  and more than 80% spontaneously beating in day 10 EBs in vector cell lines   and different switches in the two processes (S3–S6) Fig. . Moreovenduction , 49, locnduction , 51. MorThe generation of pluripotent stem cells from cloned blastocysts, especially the successful derivation of human ntESCs , holds gElf1 [Snai1/2 and Tcf3, which are reported to be involved in mesodermal commitment during ESC differentiation [We show that the global chromatin accessibility of somatic cells is successfully remodeled to ESC-like state in ntESCs. Consistent with previous studies of chromatin accessibility in SCNT embryos and iPSCs generation , 33, ourElf1 , 52, butntiation –26. The Kdm4d (H3K9me3 demethylase) can partially reduce the TAD intensity in SCNT embryos and rescue their development [Kdm4d can rescue the abnormal structures as well as development potentials in ntESCs need to be further studied. Overall, our study provides a complementary perspective to the relationship between H3K9me3 and chromatin reconstruction in the SCNT reprogramming.The 3D structures of somatic cells undergo remodeling to ESC-like structures at compartment, TAD and loop levels, but still have some abnormal chromatin structures in ntESCs Fig. . In someelopment , howeverFinally, our results show that ntESCs and iPSCs have highly similar 3D structures to each other as well as general ESCs. It is reported that somatic cell reprogramming efficiency depends on somatic cell types and methods , 55, 56.Reproducibility checklistSupplemental MaterialsTable S1Table S2Table S3Table S4Supplement video1 F35 Day10 EBSupplement video2 F40 Day10 EBSupplement video3 NT5 Day10 EBSupplement video4 NT6 Day10 EBSupplement video5 F35 Msc OE Day10 EBSupplement video6 F40 Msc OE Day10 EBSupplement video7 F35 Vetor Day10 EBSupplement video8 F40 Vetor Day10 EB


However, no related research is available about S100A9 in mouse testis. In the present study, we explored the role of S100A9 in TMs. We found that S100A9 was expressed in TMs from postnatal to adulthood and contributed to maintaining the immunosuppressive phenotype of TMs, which is associated with the activation of PI3K/Akt pathway. S100A9 treatment promotes the polarization of bone marrow-derived macrophages from M0 to M2 in vitro. S100A9 was significantly increased in TMs following UPEC-infection and elevated S100A9 contributed to maintain the M2 polarization of TMs. Treatment with S100A9 and PI3K inhibitor decreased the proportion of M2-type TMs in control and UPEC-infected mouse. Our findings reveal a crucial role of S100A9 in maintaining the immunosuppressive function of TMs through the activation of PI3K/Akt pathway, and provide a reference for further understanding the mechanism of immunosuppressive function of TMs.Macrophages are functionally plastic and can thus play different roles in various microenvironments. Testis is an immune privileged organ, and testicular macrophages (TMs) show special immunosuppressive phenotype and low response to various inflammatory stimuli. However, the underlying mechanism to maintain the immunosuppressive function of TMs remains unclear. S100A9, a small molecular Ca Polarizin vitro . Macrophin vitro .As first-line immune defense cells against inflammation in the testis, testicular macrophages (TMs) show unique tolerance to pathogens and play a key role in maintaining the immune privileged state of the testis and spermatogenesis . Tissue-2+ binding protein  mRNA level, downregulated expression of CX3CR1 protein (macrophage marker), and fewer regulatory T cells . The function of S100A9 in vitro on the polarization of bone marrow-derived macrophages was determined by exogenous supplementation of S100A9. The effect of S100A9 on the PI3K/Akt pathway was evaluated by the knockdown or overexpression of S100A9 in Raw264.7 mouse macrophage cell line. Finally, we verified that elevated S100A9 following UPEC-infection contributed to maintain the M2 polarization of TMs through the activation of PI3K/Akt pathway.In the present study, we tested the proportion of M2-type TMs in mouse from postnatal to adulthood and S100A9 expression in TMs. The effect of S100A9 on the polarization of mouse TMs was verified C57BL/6 mice   were obtained from the Animal Centre of Tongji Medical College. All animals were housed and handled in strict accordance with the guidelines approved by the Animal Care and Use Committee of Tongji Medical College, Huazhong University of Science and Technology (No. 2019_S326).E. coli (UPEC) strain CFT073 was propagated, and UPEC-induced experimental orchitis model was prepared as previously described  was dissolved in PBS. Tasquinimod  was dissolved in dimethyl sulfoxide (DMSO) and diluted with PBS to adjust the concentration of DMSO to 3%. On day 2 post-infection, ABR-215050  and LY294002 (50 mg/kg) were injected into the upper, middle and lower pole of UPEC-infected mouse testis . The concentration of S100A9 was adjusted and injected about 10-15ul into each testicle. DMSO (3%) was injected into the testis as control.Urinary pathogenic escribed . The tesThe animals were sacrificed on the 7th day after infection. Testicular tissues were collected for subsequent experiments. A small piece of testicular tissue was taken from control and UPEC-infected groups, homogenized and diluted in 1ml of sterile PBS. 100ul testicular homogenate was evenly coated on LB solid medium and incubated at 37°C overnight. The CFU of UPEC and control group was compared the next day.Total protein was extracted from macrophages by radio-immunoprecipitation assay (RIPA) buffer containing cocktail protease inhibitor and phosphor–protease inhibitor . Protein concentration was determined using BCA kit . Equal amounts of protein (5–25 µg) were loaded in appropriate concentration of sodium dodecyl sulphate (SDS)–polyacrylamide gel electrophoresis and transferred into polyvinylidene fluoride (PVDF) membrane. The membranes were blocked with 5% bovine serum albumin and incubated at 4°C overnight with the following primary antibodies: rabbit anti-S100A9 , rabbit anti-PI3K , rabbit anti-AKT , rabbit anti-p-AKT , rabbit and rabbit anti-β-actin . The membranes were then incubated with goat anti-rabbit secondary antibody  for 2 h at room temperature. Protein bands were detected using Femto-light chemiluminescence kit  and chemiluminescence-gel imaging system ECL . The results were analysed with help of the Image J software. Densitometry values were normalized to total β-actin.g for 5 min. RBC was removed with erythrocyte lysate. After resuspension with PBS, the single-cell suspension of testicular interstitial cells was obtained.The animals were sacrificed after anesthesia, and their testicles were removed. Interstitial cells were isolated as previously described . After cInterstitial cells were collected and stained according to the manufacturer’s instructions. The following antibodies were used: anti-mouse CD16/32 , Zombie-APC/CY7 , FITC anti-mouse CD45 , PE anti-mouse F4-80 , PE/cyanine5 anti-mouse CD86 , and Brilliant Violet 605™ anti-mouse CD206 . The labelled cells were detected using a flow cytometer (Cytek Aurora or BD LSRII). Data were analysed using FlowJo software .® 594 anti-mouse F4/80  as primary antibodies. The secondary antibody was goat anti-rabbit Alexa Fluor 488(1:100). The nuclei were stained with DAPI, and images were acquired using fluorescence microscope .The testicular tissue was fixed in 4% paraformaldehyde, subjected to gradient dehydration, embedded with Tissue-tek OCT , snap frozen in liquid nitrogen, and cut into 5 µm-thick sections. After antigen retrieval, the slides were permeabilized with 0.5% Tritox-100, blocked with 5% BSA, and then incubated with rabbit anti-S100A9  and Alexa FluorThe testis was fixed in 4% paraformaldehyde, embedded with paraffin wax, and cut into 5 µm-thick sections. The slides were dewaxed in xylene and rehydrated in gradient alcohol solution. After antigen retrieval, the slides were added with 3% hydrogen peroxide to prevent endogenous peroxidase formation and then blocked with 3% BSA. The expression of S100A9 was detected by incubation with primary rabbit anti-S100A9  and appropriate secondary antibodies.g for 10 min. Cellular suspension was loaded into the LS separation column previously rinsed with 3 ml of MACS running buffer and attached to MACS separator . The positive fraction (F4/80 positive macrophage) was attached to the column, while the negative fraction was eluted through the column (discarded). The magnetically labelled cells were flushed immediately by firmly pushing the plunger into the column. The cells were identified through FCM by using PE anti-mouse F4-80 . The purity of TMs was about 85%.The interstitial cells were counted, resuspended in 0.5–1 ml auto MACS running buffer , and incubated for 15 min at 4°C in the dark with Anti-F4/80-MicroBeads  according to the manufacturer’s instructions. The cells were washed and centrifuged at 300 ×®96 . The prime sequences are shown in -ΔΔCT method.The total RNA of BMDMs and TMs was extracted using TRIzol reagent  according to the manufacturer’s recommendation, and then transcribed into cDNA by using HiScript II Q RT SuperMix for qPCR . qRT-PCR amplification was performed using Hieff qPCR SYBR Green Master Mix  on LightCyclerS100A9 over-expressing vector, shRNA-expressing vector and their negative control plasmid were purchased from Genomeditech . The entire S100A9 sequence was included in the S100A9-overexpression vector. BamHI and XhoI sites were inserted in the overexpression vector PGMLV-CMV-MCS-EF1-ZsGreen1-T2A-Puro through a double-restriction enzyme digest. Three shRNAs targeting S100A9 were constructed by pGMLV-SC5 RNAi lentivirus vector. Plasmid titers were detected using Nanodrop2000  and transfected into RAW 264.7 cell lines by using High-gene Transfection reagent  according to the manufacturer’s instruction. The sequences and primers are shown in Supplementary material (All data were presented as mean and standard deviation (mean ± SD). GraphPad Prism 7  was used for data analysis through analysis of variance and Dunnett’s multiple comparisons test. P-value < 0.05 indicated significant difference.We first tested the expression of S100A9 in the testis. We found that S100A9 was expressed in the TMs of mouse from postnatal to adulthood based on F4/80 and S100A9 dual immunofluorescence localization (+CD163-(M1) and CD68+CD163+(M2). In mouse, no definite markers are available to identify TM1 and TM2; core macrophage markers such as CXC3R1, F4/80, CD11b, and AIF, are expressed in most TMs in mouse , F4/80+ (macrophage marker), CD206+ (M2 marker), and CD86+ (M1 marker). Testicular interstitial cells were collected, and the percentage of CD45+ leukocyte, CD45+F4/80+macrophage, CD45+F4/80+CD206+CD86- M2, and CD45+F4/80+CD206-CD86+ M1 groups were analysed using flow cytometry  and M2 (marked as CD45+F4/80+CD206+CD86-) in each group was detected by FCM analysis  and S100A9 into testis. DMSO was used as the control group. The percentage of M1 (marked as CD45in vitro (+F4/80+CD86+) and M2 (CD45+F4/80+CD206+), as shown in in vitro.We isolated and cultured mouse bone marrow-derived macrophages  To verify whether S100A9 can activate the immunosuppression switch of macrophages to maintain the immunosuppressive phenotype by activating PI3K/Akt pathway, we overexpressed and knocked down S100A9 in mouse RAW264.7 macrophage cell line. PI3K inhibitor (LY294002) was used to inhibit the activation of the PI3K/Akt pathway. Then, the expression levels of S100A9, PI3K, Akt, and p-Akt in the five groups, namely, the control, S100A9 knockdown (S100A9-KD), S100A9 overexpression (S100A9-OE), S100A9-KD+S100A9, and S100A9-OE+LY294002 group, were tested (We successfully established the mouse UPEC model according to previous methods . The tes+F4/80+CD86+CD206-) increased from 1.10% to 5.69%, and M2-TM (marked as CD45+F4/80+CD206+CD86-) decreased from 90% to 80.7% . Almost all adult testicular macrophages showed M2 types under physiological conditions, and this property may help in maintaining the immune-privileged state of testis to ensure that the antigens on the mature sperm surface are not attacked by themselves. S100A9 inhibitor significantly downregulated the proportion of M2-TMs from 90.0% to 58.7%, this confirming the crucial role of S100A9 in maintaining the M2 polarization state of testicular macrophages.in vitro studies of macrophage polarization because of its unchanged original “activation” status of macrophages (in vitro and increased the mRNA expression of IL-10 and TGFβ. This result is consistent with the findings in myelodysplastic syndromes (MDS)  , in whices (MDS) .in vitro, we overexpressed and knockdown S100A9 in Raw 264.7 macrophage cell line and confirmed that S100A9-OE activates the immunosuppressive switch of macrophages through the activation of PI3K/Akt pathway. Finally, we validated this phenomenon by inhibiting S100A9 and PI3K pathways in UPEC-induced orchitis. The elevated S100A9 in orchitis helps in maintaining the M2 polarization of TMs and attenuated the ratio of M1, which usually represent the ability of macrophages to resist bacteria. Similarly, the inhibition of S100A9 or PI3K pathways reduced the M2 ratio of TMs in control and UPEC group. Our results are consistent with the results of a study about fetal cord blood-derived macrophages. The high level of S100A9 in fetal cord blood altered the phenotype of fetal cord blood macrophages by preventing m-TOR expression and inhibiting glycolysis  was also decreased .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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.