In infected cells rotavirus (RV) replicates in viroplasms, cytosolic structures that require a stabilized microtubule (MT) network for their assembly, maintenance of the structure and perinuclear localization. Therefore, we hypothesized that RV could interfere with the MT-breakdown that takes place in mitosis during cell division. Using synchronized RV-permissive cells, we show that RV infection arrests the cell cycle in S/G2 phase, thus favoring replication by improving viroplasms formation, viral protein translation, and viral assembly. The arrest in S/G2 phase is independent of the host or viral strain and relies on active RV replication. RV infection causes cyclin B1 down-regulation, consistent with blocking entry into mitosis. With the aid of chemical inhibitors, the cytoskeleton network was linked to specific signaling pathways of the RV-induced cell cycle arrest. We found that upon RV infection Eg5 kinesin was delocalized from the pericentriolar region to the viroplasms. We used a MA104-Fucci system to identify three RV proteins  involved in cell cycle arrest in the S-phase. Our data indicate that there is a strong correlation between the cell cycle arrest and RV replication. Reoviridae family, is an icosahedral, non-enveloped, triple-layered particle responsible for severe diarrhea and dehydration in infants and young animals. During the RV replication cycle, receptor-mediated endocytosis and partial digestion of the infectious particle lead to the release of double-layered particles (DLPs) to the cytosol, which are transcriptionally active and initiate replication of the viral progeny , p, p45], p7v-NSP5/S67A . For infection with RV, cells were incubated for 1h at 4°C followed by incubation for the indicated time post-release at 37°C and 5% CO2.Cells were synchronized at the beginning of G1/S-phase as previously described . Briefly6 cells were centrifuged for 5 min at 530 x g. The cellular pellet was fixed with ice-cold 70% ethanol and incubated for 16 h at -20°C. The cells were centrifuged at 530 x g at 4°C for 5 min, washed once with PBS and stained in the dark for 40 min at 37°C with a 500μl propidium iodide (PI) solution . Afterward, cells were centrifuged for 5 min at 530 x g, resuspended in 1 ml PBS, filtered using a cell strainer snap-cap tube  and immediately acquired in a Gallios™ Flow Cytometer . For this purpose, 25’000 events were acquired, exciting at 488 nm with an argon laser and filter band of 695/40 nm. Data were analyzed for cell cycle using the cell cycle algorithm of FlowJo V.10.0.8r1 software (FlowJo.LLC). The cell cycle curves for DNA content were obtained using Watson or Dean-Jett-Fox (DJF) mathematical model  ratio. Values and statistical analysis were performed with Microsoft® Excel ® for Mac 2011, version 14.5.1, using an unpaired two-tailed Student’s t-test.After immunofluorescence for Eg5 detection, images were acquired using CLSM  equipped with a 63X oil objective. Data were analyzed with Leica Application Suite  and the Imaris software package . The area of distribution of Eg5 (E) and nuclei (N) were determined using ImageJ 1.42 q  and Halt™ Phosphatase Inhibitor Single-Use Cocktail (Thermo Scientific)). The cell lysates were processed as described by Eichwald l., 2004 .We previously described that RV requires the MT-network to allow the formation, maintenance and perinuclear condensation of viroplasms. Additionally, MTs become acetylated in the neighborhood of the viroplasms, a process considered as a requirement for MT-stabilization . Thus, RFollowing this line of thought, we investigated whether RV infection also arrests the cell cycle in other cell lines. Several cell lines, such as canine kidney MDCK cells, human osteosarcoma U2OS cells or green monkey fibroblast CV-1 cells were synchronized in G1/S-phase by double blocking with thymidine. Depending on the nature of the cell lines synchronized with thymidine, at 0 hpr the cell cycle histogram can be visualized with most of the cells in G1/S phase (MDCK) or G1 (U2OS and CV-1). Cells were then RV-infected, and the cellular DNA content analyzed at the indicated hours post-release . The selWe next asked whether induction of an arrest in S/G2 phase of the host cell could provide an advantage to the RV life cycle. To evaluate this, we compared RV infection in unsynchronized and synchronized MA104 cells. For this purpose, we examined NI and OSU-infected MA104 cells synchronized in G1/S phase or unsynchronized. The DNA content of NI and RV-infected cells was compared in unsynchronized and synchronized cells at 6 and 8 hpr . The celCell cycle progression is regulated and monitored by several checkpoints, verifying the phase process and repairing damaged DNA . The cheWith the aim to identify the virus life cycle step involved in cell cycle arrest, we inactivated RV virions with UV/psoralen, rendering the viral particles transcriptionally incompetent but fit for cell penetration , 58. AftTo identify host factors involved in RV cell cycle we next chose a set of drugs  that speThe drugs that had an effect on reverting the RV-arrest were the cytoskeleton inhibitors nocodazole and cytochalasin B; the allosteric inhibitor for kinesin Eg5 monastrol; the HDACs inhibitors tubacin and Na-butyrate and the CDKs inhibitor purvalanol A. All these drugs allowed progression of the cell cycle upon RV infection  suggestiet al., 2011 , 5852, 5S1 Fig(A) Immunofluorescence of non-infected (NI) and SA11-infected MA104 cells at 6 hpi. Cells were ethanol fixed for 3 min at -20°C and immunostained for viroplasms with a specific anti-NSP5 antibody (green) and nuclei stained with PI (red). Scale bar is 10μm. Representative flow cytometry histograms of synchronized MA104 cells NI and infected  with porcine OSU (B) or simian RRV (C) strains acquired at 0, 2, 4, 6, 8, 10 and 12 hpr from thymidine. (D) Immunofluorescence for viral infection of cell examined for DNA content. Each histogram overlays the DNA content by Watson mathematical model where purple, yellow and green areas under the curve correspond to the percentage values of G1, S and G2 phases, respectively. Cells were immunostained for viroplasms detection  and stained for nuclei . Scale bar is 100 μm.(TIF)Click here for additional data file.S2 Fig(A) Immunofluorescence of RRV-infected  synchronized NSP5-EGFP/MA104 cells at 6 hpr. Cells were fixed in paraformaldehyde and immunostained for cyclin B1  or PCNA , viroplasms detected with NSP5-EGFP (green) and nuclei stained with DAPI (blue). The merged image is presented in the right column. Scale bar is 10μm. (B) Flow cytometer histograms of synchronized Caco-2 cells (Human colon adenocarcinoma cells) infected with porcine OSU strain  and analyzed at 0, 2, 4, 6 and 8 hpr from thymidine. Each histogram overlays the DNA content by DJF mathematical model where purple, yellow and green areas under the curve correspond to the values of G1, S and G2 phases, respectively. (C) Plot showing the percentage of the interphase stages  from synchronized non-infected (NI) and OSU-infected Caco-2 cells at the indicated times post-release from thymidine. (D) Immunoblotting of cell lysates from OSU-infected (lanes 1 to 5) and non-infected (lanes 6 to10) synchronized Caco-2 cells. The cells were harvested at 0, 2, 4, 6 and 8 hpr. Cyclin B1, cdc2-P (Tyr 15) and NSP5 were detected using specific antibodies. GAPDH used as loading control. The molecular weight markers are indicated. (E) Images of electron microscopy of negatively stained OSU-TLPs after inactivation with UV-psoralen (UV/AMT). Scale bar is 100 nm.(TIF)Click here for additional data file.S3 FigMA104 cells were infected with OSU  and treated at 30 min post-infection with the indicated drugs. At 8 hpi, cells were fixed, immunostained for viroplasms detection  and stained for nuclei . Scale bar is 100 μm. The concentration of the drugs used was: 10μM nocodazole, 10 μM cytochalasin B, 10μM monastrol, 50μM ciliobrevin D, 100μg/ml cycloheximide, 10μM MG132, 10μM lactacystin, 10μM UBEI-41, 10μM tubacin, 5mM Na-butyrate and 10μM purvalanol A. The tested drugs, at the indicated incubation time and concentration, do not induced detectable cytotoxic effect.(TIF)Click here for additional data file.S4 FigCharacterization of synchronized MA104-Fucci cells at 0, 4 and 8 hpr from thymidine. (A) Fluorescence microscopy. Each image corresponds to the fluorescence merge from Ctd1-mKO2 (red), Geminin-mAG (green) and a bright field. The red, yellow and green arrowheads indicate the cells in early/late G1, G1/S and S/G2/M phases, respectively. Scale bar is 100μm. (B) Density plots.The cells were discriminated by its Ctd1-mKO2 (red) and Geminin-mAG (green) fluorescence intensities and gated as early G1, late G1, G1/S and S/G2/M. (C) DNA content histograms determined by PI fluorescence intensity. The data were obtained using DJF model where purple, yellow and green areas under the curve correspond to the values of G1, S and G2 phases, respectively. G1 and G2 phases were constrained. (D) Interphase stages plot . (E) Comparison plot of relative (S+G2)/G1 ratio obtained from flow cytometry of fluorescence intensities of Ctd1-mKO2 and Geminin-mAG (gray bars) or DNA content of PI fluorescence intensity (orange bars). The relative (S+G2)/G1 ratio was calculated considering NI cells at 0 hpr as a value of 1. Data represented the mean ± SEM, from three independent experiments.(TIF)Click here for additional data file.S5 Fig2 (red) and S/G2/M-mAG (green) are indicated (middle columns). A merged image is shown in the right column. Scale bar is 100 μm. (B) DNA content determined by PI fluorescence intensity. The cell cycle was calculated using DJF mathematical model where purple, yellow and green areas under the curve are the percentage values of G1, S and G2 phases, respectively. G1 and G2 phases were constrained. (C) The plot of relative (S+G2)/G1 ratio 0 and 8 hpr. (D) Immunoblotting of cell lysates of MA104 expressing RV proteins fused to CFP. The cells were transfected in the presence of VVT7.3 and lysed at 16 hpt. The membrane was incubated with anti-GFP antibody followed by the corresponding secondary antibody conjugated to peroxidase. The red dot and white arrows indicate the expected migration position for monomeric and oligomeric forms of each of the fusion proteins. The protein molecular markers are shown. The CFP tag was added at the N- or C-terminus of each RV protein as described elsewhere. Each RV protein fused to CFP was tested for functional expression by fluorescence microscopy at 24 hpt (data not shown). (E) Immunoblotting of cell lysates of MA104 expressing CFP (lane 1), NSP5-CFP wt or NSP5-CFP carrying the point mutations S67A and S67D  (lanes 2 to 4). The membrane was incubated with anti-GFP (top panel) and anti-NSP5 (bottom panel). The arrows indicate the expected migration position for NSP5-CFP. The protein molecular markers are shown. Plots for the interphase stages (%) (F) and relative (S+G2)/G1 ratio (G) of MA104-Fucci cells transfected with CFP or NSP5-CFP wt, S67A or S67D. Data were acquired at 24 hpt. The relative (S+G2)/G1 ratio was calculated using CFP only cells as value of 1. Data are represented as the mean ± SEM .Characterization of NI and OSU-infected  synchronized MA104-Fucci cells after 0 and 8 hpr from thymidine. (A) Immunofluorescence of NI (upper row) and OSU-infected (lower row) synchronized MA104-Fucci cells. Cells were immunostained at 8 hpr for viroplasms detection . Fucci sensors G1-mKO(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Anopheles gambiae immune responses mediated by the complement-like protein TEP1 against Plasmodium malaria parasites and other microbial infections. We have previously reported that the SPH CLIPA2 is a negative regulator of the TEP1-mediated response by showing that CLIPA2 knockdown (kd) enhances mosquito resistance to infections with fungi, bacteria, and Plasmodium parasites. Here, we identify another SPH, CLIPA14, as a novel regulator of mosquito immunity. We found that CLIPA14 is a hemolymph protein that is rapidly cleaved following a systemic infection. CLIPA14 kd mosquitoes elicited a potent melanization response against Plasmodium berghei ookinetes and exhibited significantly increased resistance to Plasmodium infections as well as to systemic and oral bacterial infections. The activity of the enzyme phenoloxidase, which initiates melanin biosynthesis, dramatically increased in the hemolymph of CLIPA14 kd mosquitoes in response to systemic bacterial infections. Ookinete melanization and hemolymph phenoloxidase activity were further increased after cosilencing CLIPA14 and CLIPA2, suggesting that these two SPHs act in concert to control the melanization response. Interestingly, CLIPA14 RNAi phenotypes and its infection-induced cleavage were abolished in a TEP1 loss-of-function background. Our results suggest that a complex network of SPHs functions downstream of TEP1 to regulate the melanization reaction.Clip domain serine protease homologs (SPHs) are positive and negative regulators of  Clip domain serine proteases (CLIPs)4–serine protease homologs (SPHs)), which lack one or more of the three residues  that form the protease catalytic triad. Both catalytic CLIPs and SPHs are secreted as precursor proteins and require cleavage at a specific site between the clip and protease domains to become active. The role of SPHs as inferred from studies in other insects seems to be confined to the terminal step of PPO cleavage , for the efficient cleavage and activation of PPO  identified several SPHs  to be involved in the melanization of the rodent malaria parasite Plasmodium berghei while invading the mosquito midgut epithelium , which was shown to be the active form of TEP1 that is stabilized by the two leucine-rich immune proteins APL1C and LRIM1  of these parasite stages (A); in 26% of dissected mosquitoes, midguts contained only melanized ookinetes with no single live oocyst detected. LacZ kd controls revealed a background level of melanization as expected for the G3 strain. This CLIPA14 RNAi phenotype is stronger than that observed for CLIPA2 supplemental Fig. S1). We reconstructed the full-length CLIPA14 cDNA using the GENSCAN web server at Massachusetts Institute of Technology (genes.mit.edu/GENSCAN.html)3  we used previously to silence CLIPA5  in the context of a large genetic screen of A. gambiae CLIPs . Following infection by P. berghei, CLIPA5 kd mosquitoes exhibited a basal level of ookinete melanization similar to that in LacZ kd controls (A), indicating that CLIPA5 does not regulate melanization and that its previous RNAi phenotype is due to cross-silencing CLIPA14. Indeed, Western blot analysis revealed that CLIPA5 dsRNA used in our previous genetic screen (CLIPA14 with a similar efficiency as dsCLIPA14 did (C). Despite the significant sequence similarity between CLIPA14 and CLIPA5, CLIPA14 antibody does not cross-react with CLIPA5 because CLIPA14 signal in CLIPA14/CLIPA5 double kd (dkd) was similar to that in CLIPA14 single kd mosquitoes . It is worth noting that CLIPA14 kd strongly reduces CLIPA14 hemolymph levels but does not deplete it as reported for other CLIPA kd 3  to deterae CLIPs . It is werformed . This raerformed  could becontrols A, indicac screen  silencedPA14 did C. DespitCLIPA kd , 30; howCLIPA2 kd mosquitoes also melanize a significant number of ookinetes in a TEP1-dependent manner  was 26% (B) as compared with 72 and 76% for CLIPA14 (A) and CLIPA2 (CLIPA2 was efficiently silenced by RNAi (D) as reported previously . Indeed, real-time PCR analysis revealed that dsCLIPA14 specifically silenced its target gene without affecting CLIPA2 or CLIPA5 expression levels . These results indicate that CLIPA2 and CLIPA14 exhibit additive roles in the mosquito melanization response.t manner . The RNA was 26% B as comp CLIPA14 A and CLId CLIPA2  single k by RNAi D as repoeviously . This enCLIPA14 kd mosquitoes against Plasmodium ookinetes does not necessarily indicate that these mosquitoes are similarly resistant to infections with other classes of microorganisms. This is supported by the RNAi phenotypes previously reported for the A. gambiae CTL4 gene. Although CTL4 kd mosquitoes exhibited a similar level of resistance to Plasmodium ookinetes as CLIPA14 mosquitoes  and Staphylococcus aureus (E) in their tissues at 48 h after bacterial injections as compared with controls. Interestingly, although the survival rates of E. coli-infected CLIPA14 kd mosquitoes were similar to controls , S. aureus-infected CLIPA14 kd mosquitoes showed compromised survival compared with controls  despite harboring fewer bacteria. Indeed, two criteria are known to influence the host survival to infections: reducing the microbial burden in tissues and tolerating the damage triggered by the immune response and the microbe .The potent melanotic response elicited by squitoes , they wefections . To addr E. coli D and Stas aureus E in thei microbe , 39. HenCLIPA14 kd mosquitoes are also able to clear oral infections with Serratia marcescens bacteria that are commonly present within the gut flora of field-caught mosquitoes (–CLIPA14 resulted in significantly lower numbers of S. marcescens in the hemocoel compared with LacZ kd controls (F), indicating that CLIPA14 modulates the mosquito immune response to oral bacterial infections. Also, CLIPA14 kd mosquitoes exhibited enhanced tolerance to S. marcescens infections , suggesting that Gram-negative and Gram-positive bacteria differentially influence host tolerance in this genetic background.The direct injection of bacteria into the hemocoel has been widely used as a practical approach to trigger systemic infections in mosquitoes and other insects despite that this route of infection is unlikely to be common in natural habitats due to the rigidity of the external cuticle. So we examined whether squitoes –42 and kquitoes –. Silencicontrols F, indicaCLIPA14 kd mosquitoes prompted us to address the contribution of TEP1, which is known to play an important role in antibacterial defense (E. coli colony-forming units (cfu) in LacZ (control), CLIPA14, and TEP1 single kd were compared with those in CLIPA14/TEP1 dkd mosquitoes at 48 h after bacterial injection into the hemocoel. As expected, TEP1 kd triggered increased proliferation of E. coli compared with the control group . CLIPA14 was also cleaved after E. coli injections but sometimes at a weaker level (data not shown), which prompted the use of S. aureus in these assays. The apparent molecular masses of full-length CLIPA14 (CLIPA14-F) and the cleaved C-terminal domain (CLIPA14-C) are 59 and 39 kDa, respectively. We were not able to detect the N-terminal fragment containing the clip domain, possibly because it is weakly antigenic compared with the larger C-terminal protease-like domain, hence generating an undetectable chemiluminescence signal. Of note, even CLIPA14-C band was always weak and required high exposures  for clear detection, suggesting that either a small fraction of CLIPA14-F is cleaved or the cleaved product is quickly sequestered, possibly on microbial surfaces. CLIPA14 kd strongly reduced CLIPA14 hemolymph levels by ∼85% ; this reduction is not apparent in supplemental Fig. S6A), indicating that CLIPA14 is tightly controlled by the TEP1 pathway.SPHs require proteolytic cleavage by catalytic CLIPs to become functional despite their lack of catalytic activity. This has been shown in several insect SPHs, including SPH1 and SPH2 of M. sexta , 45, PPAomphalia , and CLI gambiae . CLIPA14CLIPA14 kd triggered a potent melanotic response against Plasmodium ookinetes prompted us to ask whether these mosquitoes elicit an enhanced PO activity against systemic bacterial infections. To address this point, CLIPA14 and LacZ kd mosquitoes were injected with a suspension of live E. coli (A600 nm = 0.8), and hemolymph was extracted 3 h later to measure PO activity. Our data revealed an ∼4.5 times higher PO activity in CLIPA14 compared with that in the infected LacZ kd control (A), indeed indicating the presence of an enhanced melanotic response. CLIPA2 kd mosquitoes were also shown previously to exhibit enhanced hemolymph PO activity following E. coli infections  in CLIPA14 relative to CLIPA2 kd (B), which correlates well with the increased parasite melanization observed in the former genotype. Interestingly, the PO activity in CLIPA14/CLIPA2 dkd was even higher than that in CLIPA14 single kd, revealing an additive effect when both genes were cosilenced. Altogether, our results suggest that CLIPA2 and CLIPA14 act concertedly to regulate the TEP1-mediated immune response leading to melanization.The fact that  control A, indeedfections . Hence, LIPA2 kd B, which cut is stabilized in the mosquito hemolymph by LRIM1 and APL1C, and silencing either LRIM1 or APL1C in naïve mosquitoes triggers the loss of TEP1cut from the hemolymph , CLIPA14, TEP1, and LRIM1, and hemolymph was extracted 48 h later for Western blot analysis. As reported previously, LRIM1 kd triggered a strong reduction in TEP1cut and SPCLIP1 levels in the hemolymph; however, no effect was observed on CLIPA14, suggesting that CLIPA14 steady-state levels are not regulated by TEP1cut  mosquitoes; however, TEP1 kd did not abolish this binding, suggesting that TEP1 does not mediate CLIPA14 localization to bacterial surfaces. It is worth noting that, compared with CLIPA14, SPCLIP1 and CLIPA2 recruitment to bioparticles was more pronounced  compared with CLIPA2 , suggesting that it may be acting downstream of CLIPA2.CLIPA14 infection-induced cleavage is clearly dependent on TEP1 and its positive regulator SPCLIP1, which further supports our previous observation that SPCLIP1 acts upstream in the TEP1 pathway whereby it seems to regulate the amount of active TEP1 that is deposited on microbial surfaces . The enh of TEP1 ; howeverPlasmodium ookinete survival among the larger mosquito clip domain family, which includes several catalytic members of the CLIPB and CLIPC subgroups. For instance, although CLIPA8 . The animal protocol was approved by the Institutional Animal Care and Use Committee of the American University of Beirut (permit number 16-03-369). The Institutional Animal Care and Use Committee functions in compliance with the Public Health Service Policy on the Humane Care and Use of Laboratory Animals (United States) and adopts the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.A. gambiae G3 strain. Mosquitoes were maintained at 27 ± 0.5 °C and 80 ± 5% humidity with a 12-h day–night cycle. Larvae were reared on Tetra Goldfish food, and freshly emerged mosquitoes were collected from larval pans using a vacuum collector. Adult mosquitoes were maintained on 10% sucrose and given BALB/c mouse blood (mice were anesthetized with ketamine) for egg production. P. berghei (PbGFPCON) constitutively expressing GFP  (TEP1 (CLIPA2 (LRIM1 (CLIPA14 was produced using the following pairs of T7-tagged primers (T7 tag underlined): T7-CLIPA14-F, 5′-TAATACGACTCACTATAGGGCGGCATCATCGACATCCGTGTC-3′ and T7-CLIPA14-R, 5′-TAATACGACTCACTATAGGGGTTGCTGTCGGCGACACGCTCCT-3′. T7-tagged CLIPA14 amplicons are 326 bp in size. We have previously reported the synthesis of CLIPA5 dsRNA : T7-CLIPA5-F, 5′-TAATACGACTCACTATAGGGATTCGAGTTAATGCTGAACCTGA-3′ and T7-CLIPA5-R, 5′-TAATACGACTCACTATAGGGTGTCCATTGGACTTGATAGCATT-3′. The size of the T7-tagged CLIPA5 amplicon is 287 bp. T7-tagged amplicons were purified with GFX PCR DNA and Gel Band purification kit  and used as templates for dsRNA synthesis using the TranscriptAid T7 High Yield Transcription kit (Fermentas). Gene silencing was performed by injecting 69 nl of a 3 μg/μl solution of gene-specific dsRNA into the abdomen of each mosquito as described previously (dsRNAs for control) , TEP1 (2l)  were sorted out under a stereoscope, whereas the rest were returned back to the cups and provided with sugar pads that do not contain Serratia, and their survival was scored thereafter. A batch of at least 40 adult female mosquitoes was infected per genotype. Mosquito survival rates were scored on a daily basis over 9 days. The Kaplan–Meier survival test was used to calculate percent survival. Statistical significance of the observed differences was calculated using the log-rank test. Survival assays were repeated at least three times. cfu counts for E. coli and S. aureus were performed exactly as described previously  as a fusion with an N-terminal streptavidin tag and a C-terminal His tag coding for 10 histidines using a ligase-independent cloning (LIC) kit (Novagen) according to the manufacturer's protocol and sequenced. Because the predicted CLIPA14 coding sequence in VectorBase is partial, the sequenced full-length CLIPA14 coding sequence was deposited in GenBankTM under accession number KY344791. Production of Sf9 cells that stably express CLIPA14His and the purification of recombinant proteins from conditioned medium were performed as described previously . CLIPA14 antibody was affinity-purified over an AminoLink column (Pierce) containing covalently bound CLIPA14His according to the manufacturer's protocol.The entire eviously . PurifieLacZ (control)-, dsCLIPA14-, dsTEP1-, and dsSPCLIP1-treated mosquitoes at 1 h after intrathoracic injection of S. aureus (A600 nm = 0.8). Wild-type naïve mosquitoes were used as a control. Hemolymph protein quantification was performed using the Bradford protein assay (Fermentas), and 1.2 μg of proteins were loaded per well for all samples in a given experiment. Proteins were resolved by reducing SDS-PAGE and transferred to Immun-Blot PVDF membrane (Bio-Rad) using wet transfer (Bio-Rad). Blots were incubated with rabbit αCLIPA14 and rabbit αSRPN3  at dilutions of 1:2500 and 1:1000, respectively. Anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (Abcam) was used at 1:12,000. The experiment was repeated there times with similar results.To determine the infection-induced cleavage of CLIPA14, hemolymph was extracted from dsLRIM1 kd on CLIPA14 steady-state protein levels, hemolymph was extracted from naïve mosquitoes 48 h post-dsRNA injection. Hemolymph proteins were resolved by non-reducing SDS-PAGE, and Western blotting was performed as described above. The membrane was probed with rabbit αCLIPA14, rabbit αTEP1, rabbit αAPL1C  and subjected to Western blot analysis. The dilutions of αCLIPA14 primary and anti-rabbit IgG horseradish peroxidase-conjugated secondary antibodies were as indicated above. Efficient silencing of CLIPA2 was determined by Western blotting hemolymph extracted from naïve mosquitoes 4 days after dsCLIPA2 injection. αCLIPA2 was used at 1:1000 as described previously  using ∼5–9 μg of mosquito hemolymph per reaction as described previously  (Sigma) in a Multiskan Ex microplate reader (Thermo Labsystems). The experiment was repeated three times using different batches of mosquitoes and E. coli cultures.The PO enzymatic assay was performed 3 h after mosquito injection with eviously . The absM. A. O., N. J., and G. K. C. conceived and designed the experiments. J. N. performed the experiments. M. A. O., J. N., and G. K. C. analyzed the data. M. A. O. wrote the paper. J. N. and G. K. C. critically reviewed the manuscript.


Since the 2017 Southern Hemisphere influenza season, the A(H1N1)pdm09-like virus recommended for use in the vaccine was changed because human, but not ferret, sera distinguish A(H1N1)pdm09 viruses isolated after 2013 from the previously circulating strains. An amino acid substitution, lysine to glutamine, at position 166 (H3 numbering) in the major antigenic site of HA was reported to be responsible for the antigenic drift. Here, we obtained two anti-A(H1N1)pdm09 HA monoclonal antibodies that failed to neutralize viruses isolated after 2013 from a vaccinated volunteer. Escape mutations were identified at position 129, 165, or 166 in the major antigenic site of HA. Competitive growth of the escape mutant viruses with the wild-type virus revealed that some escape mutants possessing an amino acid substitution other than K166Q showed superior growth to that of the wild-type virus. These results suggest that in addition to the K166Q mutation that occurred in epidemic strains, other HA mutations can confer resistance to antibodies that recognize the K166 area, leading to emergence of epidemic strains with such mutations. Surveillance of circulating A(H1N1)pdm09 viruses has revealed some genetic variations in the viral surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA)4. However, until recently, the antigenicity of the circulating A(H1N1)pdm09 viruses was similar to the vaccine strain  in assays with panels of antiserum obtained from infected ferrets3. Therefore, the World Health Organization (WHO) recommended using A/California/7/2009-like virus as a vaccine seed virus until the 2016–2017 Northern hemisphere influenza season3. However, human sera distinguished the antigenicity of recent A(H1N1)pdm09 viruses from that of the A/California/7/2009-like vaccine virus, whereas ferret antisera failed to detect this antigenic difference6. Accordingly, since the 2017 Southern Hemisphere influenza season, the WHO has recommended A/Michigan/45/2015-like virus be used as the vaccine seed virus6.The first influenza pandemic of the 21st century began in 2009 with the emergence of the A(H1N1)pdm09 virus, which replaced the previous seasonal H1N1 (sH1N1) virus7. A(H1N1)pdm09-HA has five major antigenic sites, which were identified by studies using A/Puerto Rico/8/34 (H1N1)11. Two immunodominant sites (Sa and Sb) are located proximal to the receptor-binding pocket and elicit high potency neutralizing antibodies10. The Ca sites (Ca1 and Ca2) are at the subunit interface, and the Cb site is close to the stalk region of HA.10. A(H1N1)pdm09 viruses isolated after the 2012–2013 influenza season, which are classified into the genetic group 6B, possess a lysine-to-glutamine substitution at position 166  within the Sa antigenic site12. This mutation affects the antigenicity of recent A(H1N1)pdm09 viruses13. In the 2016–2017 influenza season, A(H1N1)pdm09 viruses classified into genetic group 6B.1 circulated among humans5. The 6B.1 viruses obtained a serine-to-asparagine substitution at position 165 (S165N) in the Sa antigenic site that resulted in the generation of an N-glycosylation site14.The HA protein is the major influenza viral antigen and the primary target of neutralizing antibodies17. Middle-aged adults  were reported to have a high antibody titer against the epitope around position 166 of the HA of A(H1N1)pdm09 viruses that were circulating before 2012, since they had been exposed to sH1N1 viruses that were circulating before 1985 and whose HA lacked the 129-NHT-131 glycosylation site. These middle-aged adults suffered from A(H1N)pdm09 virus infection with substantial morbidity and mortality during the 2013–2014 influenza season because of low neutralization antibody titers against the viruses possessing the K166Q substitution12. These reports demonstrate that the epitope around position 166 plays a role in the antigenic drift recently detected in assays with human, but not ferret, sera. Another group confirmed this phenomenon by using a panel of human monoclonal antibodies from a middle-aged adult17. In vitro selection of escape mutants from these monoclonal antibodies revealed that a mutation at position 166 of HA was responsible for the resistance to neutralization, suggesting that the antigenic drift was caused by the selective pressure of the human antibodies recognizing the epitope around position 16617.Previous reports have described several human monoclonal antibodies that recognize an epitope around position 166 of A(H1N1)pdm09-HA and neutralize A(H1N1)pdm09 and sH1N1 viruses. However, these antibodies failed to neutralize A(H1N1)pdm09 viruses isolated after the 2012–2013 season, which possessed the K166Q mutation, or sH1N1 viruses isolated between 1986 and 2008, which had a potential glycosylation site (129-NHT-131) masking the epitope around position 166Here, we established two human anti-A(H1N1)pdm09 HA monoclonal antibodies that recognized the epitope around position 166. We then attempted to obtain escape mutants possessing an amino acid substitution other than at position 166 and compared their growth ability with that of the wild-type virus.18. After screening by ELISA using recombinant H1-HA, hybridomas positive for anti-HA antibody production were biologically cloned. As a result of screening with 444 hybridoma clones, we obtained two monoclonal antibodies (mAbs), 1429B72/2–7 and 1429C45/1–5, which recognized A(H1N1)pdm09-HA but not H3, H5, H7, or type B-HAs. Nucleotide sequence analysis of the variable regions of each antibody  values of 0.08–0.12 and 0.24–0.39 μg/ml, respectively pdm09 viruses tested, yielding an IC50 value of 0.92–9.92 μg/ml under the same experimental conditions. These results indicate that a key amino acid in the epitope recognized by the two mAbs changed after the 2013–2014 season. To identify this key amino acid substitution, we compared the amino acid sequences of A(H1N1)pdm09 HA obtained from the Global Initiative on Sharing All Influenza Data (GISAID). As previously reported5, this analysis revealed that A(H1N1)pdm09 viruses acquired the K166Q mutation in the major antigenic site Sa after 2013 pdm09 viruses isolated in different influenza seasons. 1429B72/2-7 and 1429C45/1-5 neutralized A(H1N1)pdm09 viruses isolated between the 2009 and 2013 seasons with 50% inhibitory concentration pdm09 HA failed to neutralize sH1N1 viruses isolated between 1986 and 2008, because these viruses acquired an N-linked glycosylation at position 129, which likely covers the epitope that includes position 166. To investigate whether 1429B72/2-7 and 1429C45/1-5 have identical neutralization properties to previously reported 166-specific mAbs16, we tested the neutralization activities of our mAbs in a microneutralization assay using four sH1N1 viruses isolated in 1979, 1980, 1988, and 1992. As expected, our two monoclonal antibodies efficiently neutralized the sH1N1 viruses isolated in 1979 and 1980 but failed to neutralize the sH1N1 viruses isolated in 1988 and 1992, which contained the additional glycosylation site created by the K129N substitution, which shielded the epitope around position 166 pdm09 viruses possessing the K166Q mutation do not react with antibodies that recognize the epitope that includes position 166P < 0.01). The K166Q or K166N mutant viruses replicated to slightly higher titers compared with the wild-type virus. The S165C mutant virus replicated as efficiently as the wild-type virus, whereas the S165N mutant virus showed slightly lower titers at 12, 24, and 72 hpi. These results demonstrate that the fitness of some of the antigenic variant viruses in these human cells was increased by the escape mutation.To examine the fitness of the viruses possessing mutations that allowed them to escape from 1429B72/2-7 and 1429C45/1-5, we compared the growth kinetics of the escape mutant viruses in cultured cells with those of the wild-type virus. We infected A549 cells with the wild-type CA04 virus or a mutant virus possessing an escape mutation  or the naturally occurring HA-K166Q substitution at a multiplicity of infection (MOI) of 0.0001. Virus titers at 12, 24, 48, and 72 hours post-infection (hpi) were determined by use of plaque assays with MDCK cells Fig. . The virTo further examine the fitness of the escape mutant viruses, we performed a competitive replication assay against the wild-type virus Fig. . Briefly17, leading to the emergence of epidemic strains with such mutations.Here, we established two human anti-A(H1N1)pdm09 HA monoclonal antibodies, 1429B72/2-7 and 1429C45/1-5, which failed to neutralize A(H1N1)pdm09 viruses isolated after 2013. By passaging the CA04 virus in the presence of these monoclonal antibodies, we obtained escape mutant viruses that possessed an amino acid substitution at position 129, 165, or 166. Some escape mutants showed better growth kinetics and became the dominant population in the competitive replication assay. These findings indicate that antigenic variant viruses with a mutation at position 129 or 165 could be selected by the immunological pressure of human antibodies that recognize the epitope around position 166 that are abundant in some individuals21. The S165N substitution did not affect the antigenicity of A(H1N1)pdm09 viruses in assays with ferret anti-serum23 but no information is available in this regard with human serum. The N129D substitution led to a significant reduction in the hemagglutination inhibition (HI) titer with human sera obtained from infected or vaccinated subjects24. Although viruses with the N129D or S165N mutation were obtained in vitro and those with N129D showed high replicative ability in the competitive replication assay, the viruses with N129D did not spread in human populations. These findings suggest that the emergence of antigenic drift strains is a stochastic event in which an epidemic strain emerges randomly from viruses with similar epidemic potential, or that factors other than HA antigenicity and replicative ability in vitro  play roles in determining which antigenic variants become epidemic strains. Nonetheless, the selection of antigenic variants in vitro provides useful information for identifying future epidemic strains. Further studies are needed to understand exactly what determines which antigenic mutants became dominant in the field.Although an antigenic variant with the HA-K166Q mutation was selected in nature and became an epidemic strain in the 2012–2013 season, A(H1N1)pdm09 viruses possessing the N129D or S165N mutation temporarily appeared in the 2010–2011 season17. In these previous studies, the K166-specific antibodies were found in individuals who were born before 1979, so they had been exposed to sH1N1 viruses that circulated prior to 1985 and lacked the glycosylation site at position 12912. 1429B72/2-7 and 1429C45/1-5 were obtained from a volunteer who was born in 1976, which is consistent with this pattern. The age in humans at which antigenic drift mutants are selected in nature is not known. However, our studies, together with those of others, suggest that at least the K166Q mutants may have been selected in individuals who had been exposed to sH1N1 that circulated prior to 1985, which lacked a glycosylation site near the 166 epitope25; namely, individuals who are over 30 years old.1429B72/2-7 and 1429C45/1-5 did not neutralize A(H1N1)pdm09 isolates from after 2013 or sH1N1 isolates from after 1985 even at the highest concentration (50 μg/ml) tested. The neutralization properties of these two mAbs were similar to those of K166-specific antibodies that were reported previously50 values against A(H1N1)pdm09 viruses, these data suggest that 1429B72/2-7 may have acquired its higher neutralizing activity via these accumulated somatic mutations. Nine human monoclonal antibodies, clones 4A10, 2O10, 4K8, 6D9, 2K11, SFV009-3F05, T2-9A, T2-6A, and T2-7D, which were reported to recognize the epitope around HA-166K of A(H1N1)pdm09 and to neutralize sH1N1 viruses isolated between 1986 and 2008, utilize the VH3-7*01 gene in combination with various DH, JH, VL, and JL genes17. Although the Sa antigenic site could be recognized by human antibodies harboring various kinds of VH genes27, the VH3-7*01 gene might play an important role in developing an antibody specific for the epitope that includes position 166 of both sH1N1-HA that circulated between 1986 and 2008 and H1N1pdm09-HAs that circulated before 2013.Sequence analysis of variable heavy and light chain genes revealed that 1429B72/2-7 and 1429C45/1-5 share the VH3-7*01 gene, and somatic mutations are accumulated in 1429B72/2-7 compared with 1429C45/1-5. Together with the IC28, in vitro selection of potential antigenic drift mutants may improve the selection of vaccine seed viruses.In conclusion, amino acid substitutions other than at position 166 in major antigenic site Sa can be selected by the immunological pressure of antibodies, such as 1429B72/2-7 and 1429C45/1-5, that recognize an epitope including position 166. In combination with computational analyses and other methodsHuman blood was collected from a volunteer by following a protocol approved by the Research Ethics Review Committee of the Institute of Medical Science, the University of Tokyo , and all experiments in this manuscript were performed in accordance with the University of Tokyo’s guidelines and regulations. Written informed consent was obtained from all participants.18 were maintained in DMEM containing 15% FCS. These cells were incubated at 37 °C under 5% CO2.Madin-Darby canine kidney (MDCK) cells were maintained in Eagle’s minimal essential medium (MEM) containing 5% newborn calf serum (NCS). A549 cells were maintained in Ham’s F-12K (Kaighn’s modification) medium containing 10% fetal calf serum (FCS). Human embryonic kidney 293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FCS. SPYMEG cells29 as described below.We used the following seven A(H1N1)pdm09 viruses: A/California/04/2009 (CA04), A/Hiroshima/66/2011 (clade 5), A/Osaka/83/2011 (clade 7), A/Osaka/33/2013 (clade 6C), A/Osaka/6/2014 (clade 6B), A/Yokohama/50/2015 (clade 6B), and A/Yokohama/94/2015 (clade 6B.1), and four seasonal H1N1 viruses: A/Kumamoto/37/79, A/Kamata/8/80, A/Tokyo/913/88, and A/Minato/131/92. All of these viruses were propagated in MDCK cells. Mutant CA04 viruses were generated by reverse genetics29. Briefly, plasmids encoding mutated CA04-HA were generated by a standard PCR technique based on pPol I encoding CA04-HA. The pPol I encoding CA04-HA and the remaining 7 pPol I plasmids were cotransfected into 293T cells along with eukaryotic protein expression plasmids for PB2, PB1, PA, and NP derived from A/Puerto Rico/8/34 by use of the TransIT 293 transfection reagent (Mirus) following the manufacturer’s instructions. At 48 h post-transfection, the supernatants containing viruses were harvested and inoculated into MDCK cells. All generated viruses were sequenced to ensure the absence of unwanted mutations. Primer sequences are available upon request.Mutant viruses used in the study were generated by plasmid-based reverse genetics, as described previouslyPeripheral blood mononuclear cells (PBMCs) were isolated from a healthy volunteer, who was born in 1976 and vaccinated with the 2014–2015 seasonal influenza vaccine, by using Ficoll Paque Plus . To obtain hybridomas, the isolated PBMCs were fused with SPYMEG cells (MBL), which are fusion partner cells. The hybridomas were cultured in DMEM supplemented with 15% FCS in 96-well culture plates for 10–14 days in the presence of hypoxanthine-aminopterin-thymidine. The first screening for antibody characterization was performed by using an enzyme-linked immunosorbent assay (ELISA), as described below. Antibody-positive wells were cloned by several rounds of dilution.96-well microtiter plates were coated with 2 μg/mL of recombinant HA derived from A/California/07/2009 (H1N1pdm09), A/Perth/16/2009 (H3N2), A/Egypt/N05056/2009 (H5N1), A/Netherland/219/2003 (H7N7), B/Florida/4/2006 (Yamagata-lineage), and B/Malaysia/2506/2004 (Victoria-lineage). All recombinant HAs were purchased from Sino Biological. After being blocked with 5 times-diluted Blocking One (Nakarai), these plates were incubated with the culture medium of the hybridomas. An HRP-conjugated goat anti-human IgG, Fcγ Fragment-specific antibody (Jackson Immuno-Research) was used as a secondary antibody.http://www.ncbi.nlm.nih.gov/igblast/).Total RNA was extracted from hybridomas by using ISOGEN (Nippon gene). cDNA encoding the Fab region of a human antibody was PCR-amplified and directly sequenced. Determined nucleotide sequences of a heavy chain and a light chain were analyzed by using the IgBlast software , was performed by passaging. The human antibody in the serum-free medium was purified by using a HiTrap rProtein A FF column  and the automated chromatography system ÄKTA pure 25 .50 (Median tissue culture infectious dose) of each indicated virus in MEM containing 0.3% bovine serum albumin (BSA-MEM) were incubated with two-fold diluted antibodies (50–0.012 μg/mL) at 37 °C for 30 min. MDCK cells were washed with BSA-MEM and then incubated with the antibody-virus mixture in quadruplicate at 37 °C for 1 h. After the cells were washed twice with BSA-MEM, the cells were incubated with BSA-MEM containing 1 μg/ml L-(tosylamido-2-phenyl) ethyl chloromethyl ketone (TPCK)-treated trypsin for 3 days at 37 °C before the cytopathic effect (CPE) was examined. Antibody titers required to reduce virus replication by 50% (IC50) were calculated by using the Spearman & Kärber algorithm.To assess the neutralization capability of the antibodies, 100 TCIDAmino acid sequences of A(H1N1)pdm09 HA obtained from the Global Initiative on Sharing All Influenza Data (GISAID) were aligned. Percentages of viruses possessing the indicated amino acid at position 166 were calculated for each influenza season.Amino acid positions were plotted on the crystal structure of CA04 HA  using the PyMOL molecular graphics system to visualize the trimer.Escape mutants were selected by passaging CA04 virus in the presence of 1429B72/2-7 and 1429C45/1-5. Each two-fold diluted antibody was incubated with 10- or 100-fold diluted virus for 30 min at 37 °C. MDCK cells were washed and then incubated with the antibody-virus mixture at 37 °C for 1 h. After the inoculum was removed, the cells were incubated with BSA-MEM containing 1 μg/ml TPCK-treated trypsin for 3 days at 37 °C. Virus-containing supernatant was harvested from the CPE-positive well that contained the highest antibody concentration and was used for the next passage. We regarded a virus to be an escape mutant when it replicated well in the presence of mAbs at 50 μg/ml.Triplicate wells of confluent A549 cells were infected with the indicated viruses at a multiplicity of infection (MOI) of 0.0001, and incubated with Ham’s F-12K medium containing 0.3% BSA and 1 μg/ml TPCK-treated trypsin at 37 °C. Supernatants were harvested at 12, 24, 48, and 72 hours post-infection (hpi). Virus titers were determined by use of plaque assays with MDCK cells.The mutant CA04 viruses possessing each indicated mutation and the wild-type virus or the K166Q mutant virus were premixed at 1:1 ratio based on their PFU (plaque forming unit) titers. Triplicate wells of confluent A549 cells were infected with the virus mixtures at an estimated MOI of 0.0001, and incubated with Ham’s F-12K medium containing 0.3% BSA and 1 μg/ml TPCK-treated trypsin at 37 °C. Supernatants were harvested at 12, 24, 48, and 72 hpi. The viral RNA was isolated from each supernatant using ISOGEN LS (Nippon gene). The relative proportions of wild-type and mutant viruses in each sample were determined by using a QX200 droplet digital PCR (ddPCR) system . FAM- and HEX-labeled primer/probe mixes were designed by the Bio-Rad online service. The ddPCR reaction mixture, consisting of ddPCR Supermix (Bio-Rad), the primer/probe mix, and the template cDNA, was loaded into a cartridge with droplet generation oil (Bio-Rad), and the cartridge was placed into the droplet generator (Bio-Rad). PCR amplification was carried out using the following thermal profile: 95 °C for 10 min, followed by 40 cycles of 94 °C for 30 s and 56 °C for 1 min, 1 cycle of 98 °C for 10 min, and ending at 4 °C. After amplification, the droplets from each well of the plate were read by the droplet reader (Bio-Rad) to count the number of positive and negative droplets. ddPCR data were analyzed with QuantaSoft analysis software (Bio-Rad), and the threshold was set manually at the highest point of the negative droplet cluster. The concentration of each target gene is determined by applying a Poisson distribution calculation across all droplets, and is presented as the number of copies per μl of PCR mixture. The percentage of virus populations shows the ratio of the copy number of mutant viruses and wild-type viruses.P values < 0.01 were considered significantly different. No samples were excluded from the analysis.Statistical analysis  followed by Dunnett’s tests) was performed using GraphPad Prism software. All data analyzed during this study are included in this article.


Retrotransposons encompass half of the human genome and contribute to the formation of heterochromatin, which provides nuclear structure and regulates gene expression. Here, we asked if the human silencing hub (HUSH) complex is necessary to silence retrotransposons and whether it collaborates with TRIM28 and the chromatin remodeler ATRX at specific genomic loci. We show that the HUSH complex contributes to de novo repression and DNA methylation of an SVA retrotransposon reporter. By using naïve versus primed mouse pluripotent stem cells, we reveal a critical role for the HUSH complex in naïve cells, implicating it in programming epigenetic marks in development. Although the HUSH component FAM208A binds to endogenous retroviruses (ERVs) and long interspersed element-1s (LINE-1s or L1s), it is mainly required to repress evolutionarily young L1s (mouse-specific lineages <5 million years old). TRIM28, in contrast, is necessary to repress both ERVs and young L1s. Genes co-repressed by TRIM28 and FAM208A are evolutionarily young, or exhibit tissue-specific expression, are enriched in young L1s, and display evidence for regulation through LTR promoters. Finally, we demonstrate that the HUSH complex is also required to repress L1 elements in human cells. Overall, these data indicate that the HUSH complex and TRIM28 co-repress young retrotransposons and new genes rewired by retrotransposon noncoding DNA. Although <2% of DNA sequence in the human genome codes for proteins, the vast majority plays an enigmatic role and has thus been referred to as genomic dark matter . HoweverThe human silencing hub (HUSH) complex, composed of FAM208A , MPHOSPH8 , and PPHLN1 (periphilin 1) is recruited to genomic loci rich in H3K9me3  and inteTRIM28, in contrast, is known to silence retrotransposons early in development  and is tWe asked here if the HUSH complex, like TRIM28, is necessary for retrotransposon repression and whether it cooperates with TRIM28 and ATRX at specific genomic loci.ZNF91, but not in 293T cells . We used 293T cells for subsequent reporter assays because of their amenity to genetic manipulation.We used a human retrotransposon reporter A that is3T cells B. ReportTRIM28 knockout and complemented 293T cells. Having set up this system, we depleted the HUSH complex using shRNA before introduction of the reporter gene (MPHOSPH8 depletion (14× derepression). Although these results were obtained with single hairpins, we validated the role of Mphosph8 with an independent hairpin in mESCs . Considering that the HUSH complex is thought to function similarly to CBX proteins . Finally, we identified a role for H3F3A (4× derepression) and its ATRX–DAXX chaperone complex in establishing SVA repression .We validated that reporter repression (6.7×) C was TRIter gene D. All thter gene E, with tproteins , we deplSupplemental Fig. S1E). Of note, plasmids are chromatinized and subject to H3K9me3 . This suggested HUSH may be necessary for handover of H3K9me3 to DNA methylation and in support of this, depletion of HUSH components coincided with a decrease (5.8×) in de novo DNA methylation at the SV40 promoter along with a reduction (up to 2.7×) in SVA reporter repression . In sum, these data on the nonintegrated reporter suggest that HUSH may be required for the maintenance of retrotransposon repression.We reasoned that the HUSH complex may be targeted to the repressed SVA reporter through the chromodomain of MPHOSPH8 that interacts with H3K9me3 ; indeed, H3K9me3 . We also H3K9me3 F, as obs H3K9me3 . DNA metMphosph8, as well as Atrx and Trim28, was sufficient to reactivate retrotransposons in two different strains of serum-cultured mESCs . However, although Trim28 or Atrx depletion mainly affected intracisternal A-particle (IAP) elements, Mphosph8 depletion mainly affected L1s. Parallel culture of J1 ESCs in serum versus 2i conditions consistently led to more pronounced reactivation in the naïve cells  with HUSH components Mphosph8 and Fam208a both affecting L1 elements (up to 13× reactivation in naïve cells)  C. Deplet protein D. Finall protein E, in sup protein .Supplemental Fig. S3A) and a large proportion of TRIM28-repressed genes were co-repressed by FAM208A (94 genes), SETDB1 (183 genes), or ATRX (89 genes) . These three groups of TRIM28-repressed genes are likely direct rather than indirect targets, because up to 81% overlapped TRIM28 peaks (TRIM28-FAM208A and TRIM28-SETDB1 gene sets) and up to 77% overlapped H3K9me3 , compared to randomly-selected gene groups (“Random”) .We sought to identify genomic sites where TRIM28 collaborates with FAM208A, SETDB1, and ATRX and therefore performed mRNA-sequencing A and focRandom”) C. All thRandom”) D, and foRandom”) E, unlikeSupplemental Fig. S3D). TRIM28-SETDB1 genes were enriched for all repeats here (9× for LINEs >5 kb), whereas TRIM28-ATRX genes were enriched for full-length LTRs (4×); most interestingly, TRIM28-FAM208A genes were enriched for LINE elements of any size but particularly those >5 kb (7×)  and for satellites (2×). In fact, 81.72% of TRIM28-FAM208A genes contained an L1 or satellite or both within 20 kb (only TRIM28-regulated L1s were included) . Because TRIM28-FAM208A genes were most enriched in LINEs >5 kb , we selected all of these (within 20 kb), ordered them by their evolutionary age  and found them all to be <10% diverged from consensus sequences and the L1Md_A integrants to be the youngest by this method (mean divergence 1.53%).TRIM28 regulates genes through binding repeats . We therncluded) , and L1sncluded) G. We vernary age , and fouSupplemental Table S5). Scoring the top five repeats derepressed in each treatment group showed that IAP elements are co-repressed by TRIM28, SETDB1, and ATRX, whereas FAM208A mainly represses young L1s (from the TF and GF families) . In total, 25 families of repeats were co-repressed by TRIM28 and FAM208A  were enriched in new genes, (which we define here as mouse-specific) and tissue-specific genes. We first found 41% of genes were not conserved across placental mammals and were mouse-specific, compared to 14% of random genes . Finally, we observed that LTRs 3 kb upstream of TRIM28-FAM208A genes were biased to reside in a sense orientation (69%) suggesting they may function as promoters  to create new genes or new expression patterns , and actom genes A, left; om genes A, right.om genes B, whereaom genes B. We verromoters D.Supplemental Table S7), coating their 3′ halves . Repbase L1s bound by FAM208A (22 families with an enrichment of more than 4×) were inactive families lacking full-length copies , which clustered together (75% of ChIP-seq peaks were within 50 kb of another FAM208A peak) . FAM208A may spread to young L1s mainly from its tethering to inactive L1s and ERVs. In support of this, we found that 61% and 87% of TRIM28-FAM208A genes (that are enriched for young L1s)  D, sugges8A peak) . We coulung L1s) H containung L1s) G.Zfp180, which was bound by TRIM28 and FAM208A and at global IAP and L1 elements . This decrease in H3K9me3 was sufficient for up-regulation of Zfp180 , an increase in H3K27ac . At baseline, L1s exhibited lower levels of H3K9me3 (2.4× less), DNA methylation (3× less), and TRIM28 binding (2.5× less), suggesting that they recruit “leaky” heterochromatin and as such are readily reactivated , which recruited less H3K9me3 than TRIM28-SETDB1 genes (63% versus 77%)   C. Finallntenance F–H. L1 entenance H–J, perhpletion) G. Furthepletion) J.A key question has been whether the HUSH complex participates in retrotransposon repression and whether it collaborates with TRIM28 and its cofactors. Here, we show that the HUSH complex and TRIM28 exert nonredundant roles at evolutionarily young L1s of <5 million years old in naïve pluripotent cells. These young elements are likely prone to derepression because they bind TRIM28 and FAM208A only weakly. In the case of TRIM28, weak binding to young retrotransposons is known to result from them escaping KZNF recognition due to sequence divergence . In contIn summary, this work illustrates the complexity of epigenetic repression pathways that have evolved to regulate cellular genes through retrotransposon sequences. Future work focused on specific retrotransposon integrants will enable us to understand how these elements have been coerced to roles in gene regulation and chromatin organization in mammalian cells.129S4/SvJae) or their derived triple knockout (TKO) cells, which are knockout for Dnmt3a, Dnmt3b, and Dnmt1 (from M. Okano) were used where stated, or as an independent mESC line, ES3 ESCs  were grown in Dulbecco's Modified Eagle's Medium  high glucose with 2 mM L-Glutamine, 10% fetal calf serum (FCS), and 1% Penicillin/Streptomycin (P/S). They were split in half by cell scraping. 293T cells were grown in standard DMEM + 10% FCS and P/S. J1 ESCs  were fixed and permeabilized using intracellular staining buffers  and stained with POU5F1-PE  or isotype control  antibodies, washed, and analyzed by flow cytometry.Cells  per well in triplicate wells . Forty-eight hours post-transfection, cells were lysed, and luciferase was measured using the Dual Luciferase assay kit , an opti-plate, and a GloMax 96 microplate Luminometer (Promega) using the Dual Glow program. Raw luciferase values were normalized to Renilla luciferase values to control transfection efficiency.Dual luciferase assays were performed at ratios detailed elsewhere . 293T ceB2M and GAPDH for human genes or Cox6a1 and Gapdh for mouse genes using the −ΔΔCt method to calculate fold change. See Supplemental Table S1 for primers.Total RNA was extracted using an RNeasy mini kit (Qiagen), treated with DNase (Ambion AM1907), and 500 ng was reverse transcribed using SuperScript II (Thermo Fisher Scientific) and random primers following the manufacturer's instructions. Samples were run on an ABI 7500 Real Time PCR System (Applied Biosystems) using SYBR green Fast PCR mastermix (Life Technologies). CT values for test genes were normalized to Supplemental Table S2. Secondary antibodies were horseradish peroxidase-conjugated , and membranes were developed using ECL kits (Amersham).293T cells were washed with PBS and lysed in 2X Sample Buffer (Invitrogen) supplemented with 5% β-mercaptoethanol (Sigma). ESCs were washed 2× in ice-cold PBS and resuspended in radioimmunoprecipitation buffer with protease inhibitors added (Roche 11836170001) and quantified using the BCA Assay (Pierce). Ten micrograms was loaded per well on mini Tris/glycine gels, run in Tris/glycine/SDS buffer and mini-PROTEAN tanks (Bio-Rad) or on precast Bis-Tris gels (4%–12% or 8% gels), run in MOPs SDS buffer, followed by wet transfers to PVDF membranes. Primary antibodies are in http://bioinfo.clontech.com/rnaidesigner/sirnaSequenceDesignInit.do) and annealed and cloned into BamHI-EcoRI sites. The shRNA pLKO.1 plasmid for SETDB1 was from Miguel Branco. See Supplemental Table S3 for shRNA sequences. VSV-G-pseudotyped lentiviral vectors were produced by FuGENE 6 (Promega) cotransfection of 293T cells in 10-cm plates with 1.5 µg shRNA-encoding plasmid, 1 µg p8.91, and 1 µg pMDG2 encoding VSV-G. The harvested supernatant was used unconcentrated for cell lines or concentrated by ultracentrifugation  for primary cells.The luciferase reporter plasmids named Empty (pGL4cp-OCT4Enh-SV40), SVA_VNTR (pGL4cp-VNTR OCT4Enh E2), and L1PA4 (pGL4cp-L1PA4 OCT4Enh E2), and the human KZNF expression plasmids ZNF91 (pCAG ZNF91 HA) and ZNF93 (pCAG ZNF93) were a kind gift from David Haussler . Dual prSupplemental Methods for further details.Mouse 2i + LIF cultured J1 ESCs treated with different shRNA vectors were used for mRNA-sequencing. Cells were cultured for 5 d following puromycin selection before RNA extraction, and samples from three independent experiments were used. See https://www.girinst.org/downloads/) and the latest release downloaded (Repbase 20.06 used here). The SAMtools v.1.19 idxstats utility (https://bioconductor.org/packages/3.2/bioc/html/DESeq2.html) to identify differentially expressed repeats between samples depleted of epigenetic modifiers and controls, as previously described  procedure.Reads were mapped to rodent Repbase , and at least 10 colonies were sent for sequencing using the T7P primer. Results were analyzed using the QUMA online tool (http://quma.cdb.riken.jp) from the Riken Institute.DNA was purified with a DNeasy Blood and Tissue Kit (Qiagen), and 1 µg DNA was used for bisulfite conversion using an EpiTect Bisulfite Kit (Qiagen). Four microliters converted DNA was amplified by PCR using primers in t-tests (see figure legends for details). A P-value of <0.05 was considered statistically significant .All data in the figures are presented as the standard deviation (where there are three or more samples) or by standard error of the mean (SEM) and assessed by unpaired or paired two-tailed Student http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE107840, and processed files are also included in Supplemental Material.mRNA-sequencing and ChIP-sequencing data and processed files from this study have been submitted to the NCBI Gene Expression Omnibus (GEO;


Rheological forces in the blood trigger the unfolding of von Willebrand factor (VWF) and its A2 domain, exposing the scissile bond for proteolysis by ADAMTS13. Under quiescent conditions, the scissile bond is hidden by the folded structure due to the stabilisation provided by the structural specialisations of the VWF A2 domain, a vicinal disulphide bond, a calcium binding site and a N1574-glycan.The reduced circulating high MW multimers of VWF in patients with type 2A von Willebrand disease (VWD) may be associated with mutations within the VWF A2 domain and this is attributed to enhanced ADAMTS13 proteolysis. We investigated 11 VWF A2 domain variants identified in patients with type 2A VWD. In recombinant full-length VWF, enhanced ADAMTS13 proteolysis was detected for all of the expressed variants in the presence of urea-induced denaturation. A subset of the FLVWF variants displayed enhanced proteolysis in the absence of urea. The mechanism of enhancement was investigated using a novel VWF A2 domain FRET construct. In the absence of induced unfolding, 7/8 of the expressed mutants exhibited a disrupted domain fold, causing spatial separation of the N- and C- termini. Three of the type 2A mutants were not secreted when studied within the VWF A2 domain FRET construct. Urea denaturation revealed for all 8 secreted mutants reduced unfolding cooperativity and stability of the VWF A2 domain. As folding stability was progressively disrupted, proteolysis by ADAMTS13 increased. Due to the range of folding stabilities and wide distribution of VWF A2 domain mutations studied, we conclude that these mutations disrupt regulated folding of the VWF A2 domain. They enhance unfolding by inducing separation of N- and C-termini, thereby promoting a more open conformation that reveals its binding sites for ADAMTS13 and the scissile bond. Von Willebrand factor (VWF) is one of the largest circulating proteins in the blood, due the multimerisation/concatemerisation which occurs during its biosynthesis. VWF expression occurs exclusively in endothelial cells and megakaryocytes, with the majority of VWF in the circulation derived from endothelial cell secretion. A mature VWF monomer is formed of domains in the order D’D3-A1-A2-A3-D4-C1-C2-C3-C4-C5-C6-CK0—FRETmin)—([Fobs]t-FRETmin))/ ([Fobs]0—FRETmin). Where, [Fobs]0 is the average FRETobs value before the addition of ADAMTS13 (i.e. 0-2mins), FRETmin the calculated FRETmin value for each mutation , f(t) = FC0+FCmax*(1-exp(-t/tau)), and half-lives (tau) calculated by holding FC0 to 0 and holding FCmax to 1 (when FCmax<0.75).The VWF A2 FRET variants and recombinant ADAMTS13, 17 weremutation  and [FobWT FLVWF transiently transfected into HEK293 cells displayed a media to lysate (M:L) ratio of ~1, indicative of the regulated storage and secretion of VWF from the pseudo-WPBs formed in HEK cells–38. Of tExcept for the L1540P variant, where VWF levels were so low no protein could be detected, the majority of variants displayed a full range of VWF multimers, similar to that observed for WT FLVWF . The V14ADAMTS13 proteolysis assays were repeated in the presence of urea for these 4 uncleaved variants. After a 4 hour incubation with ADAMTS13 in the presence of 2M urea, a notable loss in multimer structure could now be observed for M1528V, V1604F and D1614G  of the N- and C- termini observed in the crystal structure, 3ZQK Fig 2A)Fig 2A). Å of the 2+ affinity chromatography, the purified proteins were examined by SDS-PAGE followed by Coomassie staining  and vice-versa (C-A2-R). To provide a positive FRET control, the VWF A2 domain was replaced with a short six amino acid linker (C-R). To provide negative FRET controls, mRuby2 or clover were removed from the expression plasmids . After estaining . Fragmenobs, the deletion variants were combined at equimolar concentrations . The increase in emission between 550-650nm with excitation at 505nm (Ex505) in the single FRET constructs, in contrast to its absence with the combination of deletion variants, indicate that FRET can be observed in the R-A2-C, C-A2-R and C-R constructs (650), further confirmed the observations made with the emission spectra  and the cooperativity of unfolding (n), as well as FRETmax and FRETmin values of the fitted curve (Urea titrations were performed to assess the influence of VWF A2 domain unfolding on the FRETed curve .Of the 11 type 2A variants introduced in the R-A2-C (in the following referred to as VWF A2 FRET) construct, three exhibited complete intracellular retention indicative of misfolding , three displayed reduced secretion in comparison to the WT  and five were secreted with similar efficiency as the WT  . Purific2+ ion, and PNGase treatment to remove the stabilising N-linked glycan from the VWF A2 domain. Selective disruption of Ca2+ binding (WT+EDTA) and removal of N-linked glycans (PNGase+CaCl2) caused a shift in Kurea of -1.40M and -0.70M in comparison to the WT (2.16±0.13M), indicating a reduction in folding stability (2+ and glycans from the WT VWF A2 FRET protein (PNGase+EDTA) resulted in a higher Kurea value (1.11±0.24) than WT+EDTA (0.77±0.03). The lower FRETmax and n values for PNGase+EDTA, in comparison to WT+EDTA and WT+CaCl2 (min). Collectively, the decrease in FRETmax, Kurea and n in the order WT+CaCl2 > PNGase+CaCl2 > WT+EDTA > PNGase+EDTA, is consistent with decrease in thermostability that we previously determined using differential scanning fluorimetry for the condition/treatment matched VWF A2 domain fragments[Control experiments were performed using EDTA to chelate the VWF A2 stabilising Catability , Table 2the WT 2.±0.13M, imax relative to the WT, 0.20±0.01 , a small reduction folding stability was observed with urea (Kurea = 1.32± 0.06M) when compared to the WT . All type 2A variants (plus EDTA and PNGase treatments of the WT) showed a statistically significant reduction in Kurea (P<0.01) using posthoc Dunnett's Multiple Comparison Test to WT CaCl2 (GraphPad Prism).Analysis of the purified type 2A VWF A2 FRET variants showed that all of the mutations, except D1653V, caused a decrease in FRET.20±0.01 . The G15.14±0.01  and disp.14±0.01 . The M15o the WT , Table 2n the WT , Table 2o the WT , Table 2obs measurements taken every 0.5 min for 120 mins. A decrease in FRETobs during the incubation with ADAMTS13 was demonstrated, with larger decreases with higher urea concentrations (obs data was converted to Fraction Cleaved (FC). The calculated FC values after a 120-minute incubation with ADAMTS13 (FC120) for the WT VWF A2 FRET are consistent with the western blot analysis of the same samples  with 50nM ADAMTS13 in comparison to 5nM ADAMTS13 . NotablyVWF gene in patients with a bleeding phenotype has been informative for understanding of the function of VWF and its domains since the 1990s[The sequencing of the the 1990s. Over 30the 1990s, while tthe 1990s. Moleculthe 1990s–33. Molethe 1990s, also, tthe 1990s, 28, shothe 1990s–13, 15.urea), the cooperativity of unfolding (n), in situ measurements allow calculation of the half-life of ADAMTS13 proteolysis and the relative proximity of the N- and C- termini of the VWF A2 domain in the absence of denaturant (see below) can be determined.To understand better the mechanism of mutations in the VWF A2 domain, we developed a VWF A2 FRET construct. Fusion of mRuby2 and clover to the N- and C- termini of the VWF A2 domain prevented aggregation and allowed the measurement of FRET between the termini and their response to urea-induced unfolding and proteolysis. Urea-induced unfolding experiments with the VWF A2 FRET construct confirmed that EDTA and PNGase treatment destabilise the VWF A2 domain, consistent with previous results using differential scanning fluorimetry (DSF), 16, 17 A representative panel of 10 mutations in the VWF A2 domain were chosen from the ISTH-SSC VWF database , together with a novel unreported variant (D1653V), . All muturea) and unfolding cooperativity (n) in comparison to the WT Fig 2D). o the WT , Table 2o the WT , a rangemax occurred for the majority of the VWF A2 variants  and fully unfolded in 6M urea , the ‘Fraction unfolded relative to WT’ can be calculated from the FRETobs values. Thus, the reduction in FRETmax of the VWF A2 variants  can be interpreted as an induced unfolding that results from the VWD mutations within the VWF A2 domain structure  disrupt the domain folding stability and cooperativity, causing spatial separation of the N- and C- termini leading to enhanced proteolysis by the regulating protease, ADAMTS13.S1 FigThe VWF A2 domain (M1574-C1670) VWD Type2A variants were transiently transfected in HEK293 EBNA cells. After 3–5 days, the media was collected, cells washed with PBS and then lysed with 1% ipegal. A 5μL aliquot of media (Med) or lysate (Lys) was dotted onto nitrocellulose membrane and left overnight. After blocking, samples were detected on a western blot using an antibody against the C-terminal Myc-tag.(PDF)Click here for additional data file.S2 Fig2.(A) Normalised emission spectra at Ex475 for 50nM and (B) excitation spectra at Em650 for 250nM R-A2-C, R-A2+A2-C, C-A2-R, C-A2+A2-R and C-R in 20mM Tris pH7.8, 50mM NaCl, 1.25mM CaCl(PDF)Click here for additional data file.S3 Fig2 and 0,2 and 6M urea. (C+D) Emission and Excitation spectra of 50nM A2-C in 20mM Tris (pH7.8), 50mM NaCl, 1.25mM CaCl2 and 0,2 and 6M urea.(A+B) Emission and Excitation spectra of 250nM R-A2 in 20mM Tris (pH7.8), 50mM NaCl, 1.25mM CaCl(PDF)Click here for additional data file.


TCF7L2) gene may be key agents in the etiology of type 2 diabetes mellitus (T2DM). In the present case-control study, we aimed to assess the possible relationship of TCF7L2 polymorphisms with T2DM and determine the effect of TCF7L2 polymorphisms on the level of fasting plasma glucose (FPG) in Eastern Chinese Han subjects. The TCF7L2 rs7903146C>T and rs290481 T>C polymorphisms were genotyped by SNPscan genotyping assays in 502 subjects with T2DM and 782 non-diabetic controls. After adjusting for age, gender, drinking, smoking and body mass index (BMI), the association of TCF7L2 rs7903146C>T and rs290481 T>C polymorphisms with T2DM was determined. We found that TCF7L2 rs290481 T>C polymorphism increased the susceptibility of T2DM in the overall comparison. In subgroup analyses by age, sex, BMI, alcohol use and smoking status, a significantly increased risk of T2DM was also found in female, older subject and never drinking and BMI < 24 kg/m2 subgroups. The relationship of TCF7L2 rs290481 T>C polymorphism with the biochemistry characteristics in controls was also assessed. We found that TCF7L2 rs290481 T>C polymorphism significantly increased the level of FPG in controls. Our findings suggest that TCF7L2 rs290481 T>C polymorphism is associated with T2DM in Eastern Chinese Han population and links to variations in FPG level. In addition, these relationships are more pronounced in female, older subject and never drinking and BMI < 24 kg/m2 subgroups. A comprehensive fine-mapping study with functional investigation is needed to confirm or refute these potential correlations.Genetic polymorphisms of the transcription factor 7-like 2 ( Type 2 diabetes mellitus (T2DM), a chronic metabolic disease, is a common form of diabetes. Over the past decades, the incidence of T2DM in Chinese adult has increased dramatically and is about 11.6%  are expressed as means ±standard deviation (SD). Comparisons between T2DM patients and healthy controls were performed with Student's t-test. The categorical variables (e.g.


The immunosuppression and immune dysregulation that follows severe injury includes type 2 immune responses manifested by elevations in interleukin (IL) 4, IL5, and IL13 early after injury. We hypothesized that IL33, an alarmin released early after tissue injury and a known regulator of type 2 immunity, contributes to the early type 2 immune responses after systemic injury.-/- mice, B6.C3(Cg)-Rorasg/sg mice deficient in group 2 innate lymphoid cells (ILC2), and C57BL/6 wild-type mice treated with anti-IL5 antibody.Blunt trauma patients admitted to the trauma intensive care unit of a level I trauma center were enrolled in an observational study that included frequent blood sampling. Dynamic changes in IL33 and soluble suppression of tumorigenicity 2 (sST2) levels were measured in the plasma and correlated with levels of the type 2 cytokines and nosocomial infection. Based on the observations in humans, mechanistic experiments were designed in a mouse model of resuscitated hemorrhagic shock and tissue trauma (HS/T). These experiments utilized wild-type C57BL/6 mice, IL33n = 472, average injury severity score [ISS] = 20.2) exhibited elevations in plasma IL33 levels upon admission and over time that correlated positively with increases in IL4, IL5, and IL13 (P < 0.0001). sST2 levels also increased after injury but in a delayed manner compared with IL33. The increases in IL33 and sST2 were significantly greater in patients that developed nosocomial infection and organ dysfunction than similarly injured patients that did not (P < 0.05). Mechanistic studies were carried out in a mouse model of HS/T that recapitulated the early increase in IL33 and delayed increase in sST2 in the plasma (P < 0.005). These studies identified a pathway where IL33 induces ILC2 activation in the lung within hours of HS/T. ILC2 IL5 up-regulation induces further IL5 expression by CXCR2+ lung neutrophils, culminating in early lung injury. The major limitations of this study are the descriptive nature of the human study component and the impact of the potential differences between human and mouse immune responses to polytrauma. Also, the studies performed did not permit us to make conclusions about the impact of IL33 on pulmonary function.Severely injured human blunt trauma patients  33, an alarmin released after tissue injury and a regulator of type 2 immunity, contributes to the early type 2 immune responses after systemic injury.We measured IL33 and its soluble receptor, soluble suppression of tumorigenicity 2 (sST2), an endogenous antagonist of IL33, in the blood of 472 multiply injured trauma patients.IL33 levels were already markedly elevated on the first blood draw after injury and correlated with increases in IL4, IL5, and IL13 as well as nosocomial infections.Reverse translation experiments using a mouse model of hemorrhagic shock and tissue trauma were carried out to understand the functional link between IL33 and the type 2 cytokine, IL5.IL33 was shown to activate a resident set of innate lymphocytes (group 2 innate lymphoid cells [ILC2]) in the lungs to produce IL5, further leading to neutrophil IL5 production.These findings demonstrate the utility of using reverse translation of observations made in humans into mechanistic experimental models that recapitulate key components of the clinical response.IL33 and one of its target cells, ILC2, are identified for the first time as regulators of the early type 2 immune responses after severe injury.The findings include a previously unsuspected role for ILC2 in the regulation of neutrophils and acute lung injury.The findings identify IL33, IL5, or their receptors as potential targets to block early lung injury after polytrauma. Severe traumatic injury is a leading cause of death and morbidity in humans between the ages of 1 and 44 years . Recent Organ failure following trauma has been linked to the magnitude of the early surge of cytokines, chemokines, and damage-associated molecular pattern (DAMP) molecules , while N+, GATA3-dependent lymphocytes found in the subepithelial space in the lungs and other barrier organs as well as adipose tissue [Tissue damage can lead to the release of self-derived “danger” signals, or alarmins, that alert the immune system to potential breeches in barrier integrity –20. IL33e tissue . ILC2 are tissue ,25. ILC2e tissue –28. An i-/- mice, and ILC2-deficient B6.C3(Cg)-Rorasg/sg mice. We tested the hypothesis that IL33 drives an early type 2 immune response in the lung through the activation of resident ILC2.In the present study, we made observations in a cohort of severely injured blunt trauma patients and reverse translated these observations in a well-described mouse polytrauma model using C57BL/6 mice, IL33http://www.equator-network.org/reporting-guidelines/strobe/  and the ARRIVE guidelines at http://www.nc3rs.org.uk/arrive-guidelines , anti-mouse CD11b FITC (M1/70), anti-mouse CD25 APC (PC61.5), anti-mouse Ly-6A/E (Sca-1) PE (D7), anti-mouse CD3e FITC (145-2C11), anti-mouse CD25 PE (PC61.5), anti-mouse CD117 PE Cy7 (ACK2), anti-mouse CD90.2 APC eFluor 780 (53–2.1), anti-mouse CD127 eFluor 450 (A7R34), anti-mouse ST2 PercP eFluor 710 (RMST2-2), anti-mouse F4/80 PE (BM8), anti-mouse CD11C PE-Cy7 (N418), anti-mouse Ly6 C Percp cy5.5 (HK1.4), anti-mouse CD11b PE-eFluor 610 (M1/70), anti-mouse CD45R PE eF610 (RA3-6B2), anti-mouse CD11b eF450 (M1/70), anti-mouse CD45.2 AF700 (104), anti-mouse CD45.1 PE-eFluor 610 (A20), anti-mouse ST2 (IL33R) APC (RMST2-2), anti-mouse CD125 BV421 (T21), OneComp eBead, and Foxp3/transcription factor staining buffer were from eBioscience. Ionomycin, PMA, nuclear staining Bisbenzimide H 33258, Histopaque 1077, and Histopaque 1191 were from Sigma. Anti-mouse CXCR2 PE (242216); immunoassay (ELISA) kit for mouse IL33, IL5, and ST2; anti-IL33 goat IgG; and recombinant mouse IL5 were from R&D Systems (P04401). Anti-mouse NK1.1 FITC (PK136); anti-mouse GATA3 PE-CF594 (L50-823) and PE-CF594 mouse IgG1, κ isotype control (X40); anti-mouse IL5 APC (TRFK5) and APC Rat IgG1, κ isotype control (R3-34); anti-mouse Ly6G APC Cy7 (1A8); anti-mouse NK1.1 APC (PK136); anti-mouse CD11b PE (M1/70); protein transport inhibitor; purified anti-mouse CD16/CD32 ; purified NA/LE anti-mouse IL5 (TRFK5); and purified NA/LE rat IgG1 κ isotype control (R3-34) were from BD Biosciences. Anti–collagen I rabbit IgG, and anti-CD45 rat IgG were from Abcam. Alexa Cy5–conjugated donkey anti-goat IgG, Alexa Cy3–conjugated donkey anti-rat IgG, and Alexa 488–conjugated donkey anti-rabbit IgG were from Jackson ImmunoResearch. Lung dissociation kit (mouse), anti-Ly6G microbead kit, and MACS LS columns were from Miltenyi Biotec. RT2 qPCR primer assay for mouse IL5 (NM_010558) and beta actin (NM_007393), Qia shredder, and RNeasy mini kit were from Qiagen. 5× iScript RT supermix and iTaq universal SYBR Green supermix were from Bio-rad. RPMI 1640 (PBS) was from Lonza Bio Whittaker. 10× cell lysis buffer was from Cell Signaling Technology. Immunoassays, MILLIPLEX map kit for human IL33, IL4, IL5, and IL13, and Luminex multiple assay were from EMD Millipore.Anti-mouse CD45 AF700 (30-F11), Blunt trauma patients presenting between the ages of 18 and 90 years old and admitted to the trauma intensive care unit were eligible for enrollment in this observational study. Written informed consent within 48 hours of presentation was obtained from all participants. Exclusion criteria included isolated head or brain injury, being unlikely to survive beyond 24 hours, and pregnancy. These criteria were chosen based on the goals of correlating early biomarker patterns with clinical outcomes over several days after systemic injury because of blunt trauma. All eligible patients were enrolled with the exception of patients presenting during time periods that research staff were not available to screen patients and obtain plasma samples. For this analysis, we also excluded 21 (out of 493) patients that died during the initial hospital stay based on the goal of measuring plasma biomarkers over a minimum of the first 7 days. The 472 blunt trauma survivors that had an average ISS of 20.2 were studied in a retrospective analysis. Plasma samples were drawn upon admission and then at 2 additional time points in the first 24 hours as well as daily for the first week. IL33 and type 2 cytokines  were measure by Luminex multiplex assay and the soluble form of ST2, soluble ST2 (sST2) by ELISA.sg/J mice, and the B6.SJL-Ptprc Pepc male mice were purchased from Jackson Laboratories. B6 IL33-/- mice were originally provided by Dr. S. Nakae [C57BL/6 male mice, B6.C3 (Cg)-RoraS. Nakae  and mainMice, age 8–12 weeks and weighing 20–30 g, were randomized into different experimental groups and anesthetized with pentobarbital sodium , with repeated doses  given when necessary during the experiment. The HS/T model was performed as previously described by our group . First, For IL5 neutralization, mice received 2 purified NA/LE anti-mouse IL5 tail vein injections (50 μg/mouse), including 30 minutes before completing the HS/T model and then immediately before the resuscitation. In the isotype control group, a purified NA/LE rat IgG1 κ isotype was injected instead.Mice were euthanized at the indicated time points after HS/T and their lungs were removed and dissociated into a single-cell suspension with a lung dissociation kit. Single cells were incubated with fixable viability dye eFluor 506 for dead cells exclusion, incubated with purified anti-mouse CD16/CD32 to block Fc receptors, and then stained with different fluorescently labeled monoclonal antibodies (mAb). For ILC2 detection (surface staining), FITC-conjugated lineage marker mAbs , anti-mouse CD45 AF700, anti-mouse CD25 APC, anti-mouse Ly-6A/E (Sca-1) PE, anti-mouse CD117 PE Cy7, anti-mouse CD90.2 APC eFluor 780, anti-mouse CD127 eFluor 450, and anti-mouse ST2 PercP eFluor 710 were used . For ILCsg/J mice were bred to use the wild-type mice (WT) (Rora+/+) and ILC2-deficienct mice (Rorasg/sg) as the BMT donors. B6.SJL-Ptprc Pepc mice were lethally irradiated at 1,000 Rads and subsequently received intravenous transplantation of 107 whole bone marrow cells from WT (Rora+/+) or Rorasg/sg mice 2–3 weeks old, were given an antibiotic diet (sulfamethoxazole and trimethoprim) for 2 weeks, and were used for the HS/T model 12 weeks after transplantation. Before making the HS/T model, about 100 μl of blood was collected from orbital sinus to identify the efficiency of BMT using the anti-mouse CD45.2 AF700 and anti-mouse CD45.1 PE-eFluor 610.Based on previous research , B6.C3. The neutrophil mRNA was extracted using RNeasy Mini Kit and RNA reverse transcribed with iScript RT supermix. PCR amplification to detect IL5 mRNA expression was performed in an iTaq Universal SYBR Green Supermix using a Bio-Rad real-time system.To assess with flow, neutrophils were isolated from mouse bone marrow as described . BrieflyFor lung IL33- and IL5-level detection, lung tissue was removed from euthanized mice at the indicated time points after HS/T. The lung protein lysis supernatant was harvested using cell lysis buffer. After the protein concentration measurement and adjustment, IL33 levels were measured by ELISA according to manufacturer’s instructions, with the protein concentration adjusted to 50 μg/sample and the protein concentration for IL5 detection at 500 μg/sample. For plasma IL33, IL5, and sST2 level detection, blood was obtained from euthanized mice by cardiac puncture and anticoagulated with heparin sodium. Plasma was collected by centrifuging the anticoagulated whole blood at 300 × g for 10 minutes at 4°C, then IL33, IL5, and soluble ST2 levels in plasma were detected by ELISA according to manufacturer’s instructions.For immunofluorescence staining, the right upper lobes of the lung were inflated, followed by perfusion fixation with PBS and then 2% paraformaldehyde. Tissue was then placed in 2% paraformaldehyde for an additional 2 hours and then switched to 30% sucrose in a distilled water solution for 12 hours. The tissue was then slowly frozen in 2-methylbutane according to a standardized protocol for cryopreservation . Tissue Right upper lung lobes processed as described above were stained for hematoxylin and eosin (H&E) to evaluate histopathologic cumulative changes among treatment groups . Images t test. Group–time interactions of plasma inflammatory mediator levels in patient groups were compared by 2-way ANOVA. Correlations were measured with the pairwise Pearson correlations test using GraphPad Prism 6 software . In all studies, differences were considered significant when P < 0.05.The results presented in the study are expressed as the mean ± SEM. Statistical differences between the 2 groups were established by unpaired 2-tailed Student http://dx.doi.org/10.5061/dryad.kt8m4 [Data were deposited in the Dryad repository: ad.kt8m4 .n = 12), trauma patients exhibited significant elevations in IL33 levels at admission (n = 12) had an average baseline IL33 level of 22.8 pg/mL. This is midrange (5–53 pg/mL) for what has been reported in healthy adult humans [IL33 is described as an alarmin that is released after tissue injury; however, the patterns of IL33 release in humans experiencing multiple trauma have not been determined. We measured circulating IL33 levels over time in 472 blunt trauma patients admitted to our trauma intensive care unit (ICU). The demographic and clinical characteristics of these patients have been described previously in detail (7). Briefly, the subjects had an average age of 42 (range 18 to 90), were ~70% male, and had an average ISS of 20.2 (range 1 to 50). Blood samples were obtained upon admission, at 2 additional time points in the first 24 hours, and then daily until day 7 or discharge from the ICU. Compared to IL33 levels obtained in healthy volunteers  matched for injury severity (based on ISS), age, and sex to exclude potential confounding based on differences in these variables . Taken together, the human analyses show that IL33 is elevated in severely injured humans as early as the time of presentation to a trauma center and that elevations in circulating IL33 correlated with complications and circulating type 2 cytokine levels.Using multivariable regression analyses controlling for age, sex, or ISS, we found similar significant correlations between IL33 and type 2 cytokines independent of these variables  cells peaked at 6 hours (P < 0.05) and then declined gradually (+ cells that were neutrophils (Ly6G+CD11b+ gated from CD45+ cells) increased after HS/T and also peaked at 6 hours (P < 0.05). The percentage of T cells (CD45+CD3e+), B cells (CD45+CD45R+), and natural killer (NK) cells (CD45+NK1.1+) gradually decreased in the lungs of C57BL/6 mice after HS/T  to establish changes in percentage of ILC2 or quantitate changes in IL5 expression by combining surface staining for CD45+Lin-CD25+CD127+ with intracellular staining for GATA3+ and IL5. We found the frequency of ILC2 increased in the lungs of C57BL/6 mice after HS/T. This increase could be detected at 6 hours and increased further at 24 hours (P < 0.05)  . Thus, I-/- mice to HS/T and found that both the increase in ILC2 frequency and intracellular ILC2 IL5 expression at 6 hours after HS/T were dependent on IL33 (P < 0.05). Whereas the frequency of neutrophils in the lungs after HS/T was not influenced by IL33 deletion (+ cells gated from CD45+Ly6G+CD11b+) (P < 0.05)  . ImmunohL/6 mice . This inTwo epithelial cytokines other than IL33, IL25, and thymic stromal lymphopoietin (TSLP) are known to activate ILC2 in the lung ,24. Althsg/sg mice into lethally irradiated B6.SJL-Ptprc Pepc mice as described by others [sg/sg) mice at 12 weeks (P < 0.05), and no increase in ILC2 was observed in the lungs of the ILC2-deficient mice at 6 hours or 24 hours after HS/T (P < 0.05) (ILC2-deficient mice were created by adoptive transfer of bone marrow from Roray others . We confter HS/T . Deletioter HS/T  but abla < 0.05) . Thus, uIt is not feasible to isolate lung tissue for assessment of ILC2 populations in the lung after severe trauma in humans. Instead, we sought to establish evidence if ILC2 are engaged early after injury in humans. Circulating leukocytes were isolated from 4 severely injured blunt trauma patients upon admission and at 24 and 48 hours after admission. The numbers of ILC2 in the blood of the injured subjects were compared to the numbers in 3 healthy control subjects. The absolute counts of ILC2 were significantly lower in the trauma patients even at the time of the initial blood draw and remained lower for the first 48 hours after injury . We hypoP < 0.05) (P < 0.05)  . As addi < 0.05) . More im < 0.05)  but abla < 0.05) . In totaP < 0.05)  . In cont < 0.05) . Thus, tRecent findings indicate that IL33-stimulated ILC2 are a major source of type 2 cytokines in the lung, where these resident innate lymphocytes potently regulate immune responses ,26,59,60The roles of type 2 immune responses depend on the tissue type and are important for antihelminth immunity, metabolic homeostasis, allergic inflammation, and reparative processes ,61,62. TAfter injury, those patients prone to complications, such as NI and MODS, have elevated type 2 cytokines in their circulation . A polarIL33 is a member of the IL1 cytokine superfamily and functions as an alarmin when released from damaged or necrotic cells . Membran+ cells. ILC2 are typically present in low numbers; however, the percentage of CD45+ cells that were ILC2 in the lung increased rapidly after systemic injury. Despite their low numbers, these noncytolytic, regulatory innate lymphoid cells are a potent source of type 2 cytokines [Although IL33 can regulate type 2 responses through multiple cellular targets, we found that elevations of IL5 in the lung were entirely dependent on ILC2. In fact, the frequency of lymphocytes dropped in the lung after HS/T, and this included CD4ytokines ,26,68. I- and emerge in response to IL25 [Other epithelial cytokines, including IL25 and TSLP, also regulate ILC2 function. We found that IL25 and TSLP were elevated in the plasma of mice subjected to HS/T, and both were present at baseline in the lungs. Neutralizing antibodies delivered to either IL25 or TSLP did not alter the production of IL5 by ILC2 or neutrophils after HS/T  Figs. Re to IL25 . However+ neutrophils were the major source of IL5 in the lung after HS/T. Neutrophils are ST2-negative, which led us to show that neutrophils express the IL5 receptor and can respond to IL5 to produce their own IL5. Thus, we conclude that one of the important effects of IL33 in the lung after systemic shock and injury is the rapid release of IL5 by ILC2, which leads to more IL5 production by neutrophils that have been attracted to the lung as the result of increased CXCR2 expression. IL5 can stimulate neutrophil H2O2 release [Other than ILC2, we found that CXCR2 release ,71, whic+ neutrophils expressed more IL5 than CXCR2- neutrophils after HS/T. Others have shown that CXCR2+ is important for neutrophils to migrate to sites of inflammation [We found that CXCR2ammation . Deletioammation . IL33-inammation . We did The strengths of this study include conclusive data on the correlation of IL33 with type 2 cytokines in an adequately powered study of injured humans as well as the definitive mechanistic studies in a mouse polytrauma model that show IL33 activates ILC2 in the lungs, leading to neutrophil IL5 production. However, there are important limitations. The observations in humans are only correlations. The degree to which the mechanistic studies in mice translate back to the human condition is uncertain. Because there is a preponderance of male severely injured patients, we chose to include only male mice in this initial study. Future studies should include a comparison between males and females. Finally, we did not show whether the histopathologic changes observed in the lungs of mice correlate with changes in pulmonary function.In summary, our studies demonstrate how reverse translation of human observations into mechanistic experimental models can be used to gain insights into trauma-induced immune dysfunction. This strategy allowed us to identify an unsuspected role for IL33-mediated activation of ILC2 in the initial inflammatory response in the lung after systemic injury. ILC2 regulate neutrophil function through IL5, which promotes IL5 expression in neutrophils and drives lung inflammatory changes. As lung dysfunction is a common and serious problem in patients experiencing hemorrhagic shock and tissue trauma, the findings may have identified a novel contributing pathway to this pathologic process that could be targeted through blocking IL33, IL5, or their receptors.S1 FigN = 335. Correlation coefficient: –0.13, P = 0.015.(TIF)Click here for additional data file.S2 FigThere were no differences in the average levels between female and male subjects.(TIF)Click here for additional data file.S3 Fig+Siglec-F+ cells; no increase is shown after HS/T.Eosinophils in the lungs were evaluated by gating on CD45(TIF)Click here for additional data file.S4 Fig-/- mice at 6 hours after HS/T, like the up-regulation in the WT C57BL/6 mice. Red, neutrophil; blue, nuclei; green, actin. 20 x 2z magnification, scale bar 50 μm.Immunohistochemistry of Ly6G also confirmed that neutrophils were up-regulated in the lungs of IL33(TIF)Click here for additional data file.S5 Fign = 4–6/group, by ELISA). B: IL25 in the lungs maintained at a constant level after different time points after HS/T . C: IL25 neutralization antibody showed no obvious effects on the increased neutrophil IL5 or ILC2 IL5 expression after HS/T (n = 2–5/group). * P < 0.05.A: The IL25 level in plasma was increased after HS/T, especially at 6 hours ((TIF)Click here for additional data file.S6 Fign = 4–8/group, by ELISA). B: TSLP level did not show obvious change in the lungs after HS/T. C: Neutralizing antibody delivered to TSLP did not show obvious effects on the increased neutrophil IL5 or ILC2 IL5 expression after HS/T (n = 2–5/group). * P < 0.05.A: TSLP increased gradually after HS/T in plasma ((TIF)Click here for additional data file.S7 Fig+, CRTH2+. The absolute counts of ILC2 were significantly lower in the trauma patients even at the time of the initial blood draw and remained lower for the first 48 hours after injury. Controls, n = 3; patients, n = 5. Cell number x 103/ml of blood is depicted. * P < 0.05.ILC2 in peripheral blood leukocytes (PBL) were evaluated by gating on CD3 negative and lineage-negative cells, followed by gating on CD127(TIF)Click here for additional data file.S1 TableP < 0.05.Values are expressed as mean ± SEM. Mann–Whitney U-Test and Fisher’s exact test were used as appropriate with statistical significance set at (DOCX)Click here for additional data file.S1 Text(DOC)Click here for additional data file.S2 TextThe prospective analysis plan was submitted as part of our IRB application on November 2015.(DOCX)Click here for additional data file.S3 Text(DOCX)Click here for additional data file.